Production of lysosomal enzymes in plants by transient expression

ABSTRACT

The invention relates to α-galactosidase truncated at the carboxy terminus and the production of enzymatically active recombinant human and animal lysosomal enzymes involving construction and expression of recombinant expression constructs comprising coding sequences of human or animal lysosomal enzymes in a plant expression system. The plant expression system provides for post-translational modification and processing to produce a recombinant gene product exhibiting enzymatic activity. The invention is demonstrated by working examples in which transgenic tobacco plants express recombinant expression constructs comprising human glucocerebrosidase nucleotide sequences. The invention is also demonstrated by working examples in which transfected tobacco plants express recombinant viral expression constructs comprising human α galactosidase nucleotide sequences. The recombinant lysosomal enzymes produced in accordance with the invention may be used for a variety of purposes, including but not limited to enzyme replacement therapy for the therapeutic treatment of human and animal lysosomal storage diseases.

RELATED APPLICATIONS

This present application is a continuation-in-part of U.S. applicationSer. No. 09/626,127, filed Jul. 26, 2000.

FIELD OF THE INVENTION

This invention is in the field of therapeutic peptides. Specificallythis invention relates to the production of pharmaceutical peptides andproteins encoded on a recombinant plant virus or and produced by aninfected plant or produced by a recombinant plant. The present inventionrelates especially to the production of human and animal lysosomalenzymes in plants comprising expressing the genetic coding sequence of ahuman or animal lysosomal enzyme in a plant expression system. The plantexpression system provides for post-translational modification andprocessing to produce recombinant protein having enzymatic activity. Theinvention is demonstrated herein by working examples in which transgenicor transfected tobacco plants produce a modified human α galactosidaseand a glucocerebrosidase, both of which are enzymatically active. Therecombinant lysosomal enzymes produced in accordance with the inventionmay be used for a variety of purposes including but not limited toenzyme replacement therapy for the therapeutic treatment of lysosomalstorage diseases, research for development of new approaches to medicaltreatment of lysosomal storage diseases, and industrial processesinvolving enzymatic substrate hydrolysis.

BACKGROUND OF THE INVENTION

Lysosomes, which are present in all animal cells, are acidic cytoplasmicorganelles that contain an assortment of hydrolytic enzymes. Theseenzymes function in the degradation of internalized and endogenousmacromolecular substrates. When there is a lysosomal enzyme deficiency,the deficient enzyme's undegraded substrates gradually accumulate withinthe lysosomes causing a progressive increase in the size and number ofthese organelles within the cell. This accumulation within the celleventually leads to malfunction of the organ and to the gross pathologyof a lysosomal storage disease, with the particular disease depending onthe particular enzyme deficiency. More than thirty distinct, inheritedlysosomal storage diseases have been characterized in humans.

Enzyme Replacement Therapy

One proven treatment for lysosomal storage diseases is enzymereplacement therapy in which an active form of the enzyme isadministered directly to the patient. However, abundant, inexpensive andsafe supplies of therapeutic lysosomal enzymes are not commerciallyavailable for the treatment of any of the lysosomal storage diseases.There are a large number of metabolic storage disorders known to affectman. As a group, these diseases are the most prevalent geneticabnormalities of humans, yet individually they are quite rare. One ofthe three major classes of these conditions, comprising the majority ofpatients, is the sphingolipidoses in which excessive quantities ofundegraded fatty components of cell membranes accumulate because ofinherited deficiencies of specific catabolic enzymes. Principaldisorders in this category are Gaucher disease, Niemann-Pick disease,Fabry disease, and Tay-Sachs disease. All of these disorders are causedby harmful mutations in the genes that code for specific housekeepingenzymes within lysosomes. Thus, to be effective, enzyme replacementtherapy requires that the requisite exogenous enzyme be taken up by thecells in which the materials are catabolized and that they beincorporated into lysosomes within these cells. Fabry disease is anideal candidate for enzyme replacement therapy because the disease doesnot involve the central nervous system. The therapeutic enzyme does notneed to be delivered across the blood-brain barrier (1, 2).

The effectiveness of enzyme replacement therapy has been dramaticallydocumented in the treatment of patients with Gaucher disease. Thiscondition is the most frequent of all metabolic storage disorders. It isestimated that there are 15,000 patients with this condition in theUnited States and about 80,000 worldwide. Soon after the enzymaticdefect in Gaucher disease was established, consideration was given tothe possibility of treating patients with purified α-glucocerebrosidase(3). Dr. Brady elected to use human placental tissue as the source ofenzyme in order to minimize sensitizing patients to the exogenousprotein. Initial studies with small amounts of glucocerebrosidaseinjected intravenously into patients with Gaucher disease revealed thatthe exogenous enzyme reduced the quantity of accumulatedglucocerebroside in the liver and in the blood (4). A large-scale enzymepurification procedure was developed in order to obtain sufficientquantities for clinical efficacy trials (5). It was then learned thatmodifications of the terminal sugars on oligosaccharide chains of theenzyme were necessary in order to target intravenously administeredenzyme to macrophages where most of the glucocerebroside is stored.Targeting to macrophages was accomplished by sequential enzymaticremoval of monosaccharide residues from glucocerebrosidase resulting inmannose-terminal glucocerebrosidase (6). Administration of thisglycoform of glucocerebrosidase to patients has brought about immenseimprovement in their condition (7-10). The modified enzyme (alglucerase)is now produced commercially by Genzyme Corporation in Cambridge, Mass.,under the trade name Ceredase™. The beneficial effects of this treatmenthave been universally confirmed (11-13). Production of recombinantglucocerebrosidase (imiglucerase) is underway in Chinese hamster ovary(CHO) cells, and the product (Cerezyme™) is as effective as placentalglucocerebrosidase (14). The experience with Gaucher treatment validatesenzyme replacement therapy with a product that requirespost-translational modifications.

Fabry disease is caused by deficiencies in the catalytic activity of thelysosomal enzyme α galactosidase A (Gal-A). Human Gal-A is aglycoprotein homodimer with a molecular weight of approximately 101 kDacontaining 5-15% Asn-linked carbohydrate. The enzyme containsapproximately equal portions of high mannose and complex type glycans.Upon isoelectric focusing, many forms of the enzyme are observed due todifferences in sialylation depending on the source of the protein(tissue or plasma forms). The disease is inherited as an X-linkedrecessive trait. A number of specific mutations in the gene have beencharacterized, including partial rearrangements, splice-junction defectsand point mutations. Most of these mutations are private and therefore,the gene appears to be highly mutable relative to genes encoding otherhousekeeping enzymes. Defects result in the accumulation ofglycosphingolipid substrates, globotriaosylceramide and relatedglycolipids with terminal α-galactosidic linkages. Uncatabolizedsubstrate accumulates in the plasma, vascular endothelium and variousorgans leading to an early demise from vascular disease of the heart,brain, and kidney, particularly in the classically affected hemizygousmales. In addition to systemic disease, affected individuals oftensuffer from peripheral neuropathies and have characteristicangiokeratoma of the skin. Heterozygous female carriers may have a moreattenuated range of disease phenotypes (1,2).

Exploratory trials of enzyme replacement therapy for Fabry disease havedemonstrated the biochemical effectiveness of this approach (15-18).Repeated injections of purified splenic and plasma Gal-A reduced thelevel of plasma substrate and may have mobilized stored tissue substrateinto circulation. No immunological complications were apparent inrepeated infusions of enzyme into hemizygous males. Furtherinvestigations have not been attempted because of the great difficultyin obtaining sufficient quantities of enzyme for a meaningfulreplacement trial. The availability of large quantities of enzyme wouldenable optimization of glycoforms for therapeutic efficacy by improvingcell targeting and prolonging the half-life in circulation and targetorgans.

α Galactosidase

In the early 1970's, several investigators demonstrated the existence oftwo .α.-Galactosidase isozymes designated A and B, which hydrolyzed theα-galactosidic linkages in 4-MU-and/or rho-NP-α-D-galactopyranosides(62, 63, 64, 65, 66, 67, 68, 69) In tissues, about 80%-90% of totalα-Galactosidase (α-Gal) activity was due to a thermolabile,myoinositol-inhibitable α-Gal A isozyme, while a relativelythermostable, α-Gal B, accounted for the remainder. The two “isozymes”were separable by electrophoresis, isoelectric focusing, and ionexchange chromatography. After neuraminidase treatment, theelectrophoretic migrations and pI value of α-Gal A and B were similar(70), initially suggesting that the two enzymes were the differentiallyglycosylated products of the same gene. The finding that the purifiedglycoprotein enzymes had similar physical properties including subunitmolecular weight (about 46 kDa), homodimeric structures, and amino acidcompositions also indicated their structural relatedness (70. 71. 72.73. 74. 75. 76. 77). However, the subsequent demonstration thatpolyclonal antibodies against α-Gal A or B did not cross-react with theother enzyme (78, 79) that only α-Gal A activity was deficient inhemizygotes with Fabry disease (80, 81, 82, 83, 84, 85, 86) and that thegenes for α-Gal A and B mapped to different chromosomes (Desnick, etal., 1989, in The Metabolic Basis of Inherited Disease, Scriver, C. R.,Beaudet, A. L. Sly, W. S. and Valle, D., eds, pp. 1751-1796, McGrawHill, New York; deGroot, et al., 1978, Hum. Genet. 44:305-312), clearlydemonstrated that these enzymes were genetically distinct.

α-Gal A and Fabry Disease

In Fabry disease, a lysosomal storage disease resulting from thedeficient activity of α-Gal A, identification of the enzymatic defect in1967 (Brady, et al., 1967, N. Eng. J. Med. 276:1163) led to the first invitro (Dawson, et al., 1973, Pediat. Res. 7: 694-690m) and in vivo(Mapes, et al., 1970, Science 169:987) therapeutic trials of α-Gal Areplacement in 1969 and 1970, respectively. These and subsequent trials(Mapes, et al., 1970, Science 169:987; Desnick, et al., 1979, Proc.Natl. Acad. Sci. USA 76: 5326; and, Brady, et al., 1973, N. Engl. J.Med. 289: 9) demonstrated the biochemical effectiveness of direct enzymereplacement for this disease. Repeated injections of purified splenicand plasma α-Gal A (100,000 U/injection) were administered to affectedhemizygotes over a four month period (Desnick, et al., 1979, Proc. Natl.Acad. Sci. USA 76:5326). The results of these studies demonstrated that(a) the plasma clearance of the splenic form was 7 times faster thanthat of the plasma form (10 min vs 70 min); (b) compared to the splenicform of the enzyme, the plasma form effected a 25-fold greater depletionof plasma substrate over a markedly longer period (48 hours vs 1 hour);(c) there was no evidence of an immunologic response to six doses ofeither form, administered intravenously over a four month period to twoaffected hemizygotes; and (d) suggestive evidence was obtainedindicating that stored tissue substrate was mobilized into thecirculation following depletion by the plasma form, but not by thesplenic form of the enzyme. Thus, the administered enzyme not onlydepleted the substrate from the circulation (a major site ofaccumulation), but also possibly mobilized the previously storedsubstrate from other depots into the circulation for subsequentclearance. These studies indicated the potential for eliminating, orsignificantly reducing, the pathological glycolipid storage by repeatedenzyme replacement. However, the biochemical and clinical effectivenessof enzyme replacement in Fabry disease has not been commerciallyavailable due to the lack of sufficient human enzyme for adequate dosesand longterm evaluation.

The α-Gal A Enzyme

The α-Gal A human enzyme has a molecular weight of approximately 101,000Da. On SDS gel electrophoresis it migrates as a single band ofapproximately 49,000 Da indicating the enzyme is a homodimer (Bishop &Desnick, 1981, J. Biol. Chem. 256:1307). α-Gal A is synthesized as a50,500 Da precursor containing phosphorylated endoglycosidase Hsensitive oligosaccharides. This precursor is processed to a mature formof about 46,000 Da within 3-7 days after its synthesis. Theintermediates of this processing have not been defined (Lemansky, etal., 1987, J. Biol. Chem. 262:2062). As with many lysosomal enzymes,.α.-Gal A is targeted to the lysosome via the mannose-6-phosphatereceptor. This is evidenced by the high secretion rate of this enzyme inmucolipidosis II cells and in fibroblasts treated with NH.sub.4 Cl.

The enzyme has been shown to contain 5-15% Asn linked carbohydrate(Ledonne, et al., 1983, Arch. Biochem. Biophys. 224:186). The tissueform of this enzyme was shown to have about 52% high mannose and 48%complex type oligosaccharides. The high mannose type coeluted, onBio-gel chromatography, with Man.sub.8-9 GlcNAc while the complex typeoligosaccharides were of two categories containing 14 and 19-39 glucoseunits. Upon isoelectric focusing many forms of this enzyme are observeddepending on the sources of the purified enzyme (tissue vs plasma form).However, upon treatment with neuraminidase, a single band is observed(pI-5.1) indicating that this heterogeneity is due to different degreesof sialylation (Bishop & Desnick, 1981, J. Biol. Chem. 256:1307).Initial efforts to express the full-length cDNA encoding α-Gal Ainvolved using various prokaryotic expression vectors (Hantzopoulos andCalhoun, 1987, Gene 57:159; Ioannou, 1990, Ph.D. Thesis, City Universityof New York). Although microbial expression was achieved, as evidencedby enzyme assays of intact E. coli cells and growth on melibiose as thecarbon source, the human protein was expressed at low levels and couldnot be purified from the bacteria. These results indicate that therecombinant enzyme was unstable due to the lack of normal glycosylationand/or the presence of endogenous cytoplasmic or periplasmic proteases.

Gaucher Disease and Treatment

Gaucher disease is the most common lysosomal storage disease in humans,with the highest frequency encountered in the Ashkenazi Jewishpopulation. About 5,000 to 10,000 people in the United States areafflicted with this disease (Grabowski, 1993, Adv. Hum. Genet.21:377-441). Gaucher disease results from a deficiency inglucocerebrosidase (hGCB); glucosylceramidase; acid β-glucosidase; EC3.2.1.45). This deficiency leads to an accumulation of the enzyme'ssubstrate, glucocerebroside, in reticuloendothelial cells of the bonemarrow, spleen and liver, resulting in significant skeletalcomplications such as bone marrow expansion and bone deterioration, andalso hypersplenism, hepatomegaly, thrombocytopenia, anemia and lungcomplications (Grabowski, 1993, supra; Lee, 1982, Prog. Clin. Biol. Res.95:177-217; Brady et al., 1965, Biochem. Biophys. Res. Comm.18:221-225). hGCB replacement therapy has revolutionized the medicalcare and management of Gaucher disease, leading to significantimprovement in the quality of life of many Gaucher patients (Pastores etal., 1993, Blood 82:408-416; Fallet et al., 1992, Pediatr. Res.31:496-502). Studies have shown that regular, intravenous administrationof specifically modified hGCB (Ceredase.™., Genzyme Corp.) can result indramatic improvements and even reversals in the hepatic, splenic andhematologic manifestations of the disease (Pastores et al., 1993, supra;Fallet: et al., 1992, supra; Figueroa et al., 1992, N. Eng. J. Med327:1632-1636; Barton et al., 1991, N. Eng. J. Med. 324:1464-1470;Beutler et al., 1991, Blood 78:1183-1189). Improvements in associatedskeletal and lung complications are possible, but require larger dosesof enzyme over longer periods of time.

Despite the benefits of hGCB replacement therapy, the source and highcost of the enzyme seriously restricts its availability. Until recently,the only commercial source of purified hGCB has been from pooled humanplacentae, where ten to twenty kilograms (kg) of placentae yield only 1milligram (mg) of enzyme. From five hundred to two thousand kilograms ofplacenta (equivalent to 2,000-8,000 placentae) are required to treateach patient every two weeks. Current costs for hGCB replacement therapyrange from $55 to $220/kg patient body weight every two weeks, or from$70,000 to $300,000/year for a 50 kg patient. Since the need for therapyessentially lasts for the duration of a patient's life, costs for theenzyme alone may exceed $15,000,000 during 30 to 70 years of therapy.

A second major problem associated with treating Gaucher patients withglucocerebrosidase isolated from human tissue (and perhaps even fromother animal tissues) is the risk of exposing patients to infectiousagents which may be present in the pooled placentae, e.g., humanimmuno-deficiency virus (HIV), hepatitis viruses, and others.

Accordingly, a new source of hGCB is needed to effectively reduce thecost of treatment and to eliminate the risk of exposing Gaucher patientsto infectious agents.

Hurler Syndrome and Treatment

Hurler syndrome is the most common of the group of human lysosomalstorage disorders known as the mucopolysaccharidosis (MPS) involving aninability to degrade dermatan sulfate and heparan sulfate. Hurlerpatients are deficient in the lysosomal enzyme, α-L-iduronidase (IDUA),and the resulting accumulation of glucosaminoglycans in the lysosomes ofaffected cells leads to a variety of clinical manifestations (Neufeld &Ash well, 1980, The Biochemistry of Glycoproteins and Proteoglycans, ed.W. J. Lennarz, Plenum Press, N.Y.; pp. 241-266) including developmentaldelay, enlargement of the liver and spleen, skeletal abnormalities,mental retardation, coarsened facial features, corneal clouding, andrespiratory and cardiovascular involvement. Hurler/Scheie syndrome (MPSI H/S) and Scheie syndrome (MPS IS) represent less severe forms of thedisorder but also involve deficiencies in IDUA. Molecular studies on thegenes and cDNAs of MPS I patients has led to an emerging understandingof genotype and clinical phenotype (Scott et al., 1990, Am. J. Hum.Genet. 47:802-807). In addition, both a canine and feline form of MPS Ihave been characterized (Haskins et al., 1979, Pediat. Res.13:1294-1297; Haskins and Kakkis, 1995, Am. J. Hum. Genet. 57:A39 Abstr.194; Shull et al., 1994, Proc. Natl. Acad. Sci. USA, 91:12937-12941)providing an effective in vivo model for testing therapeutic approaches.

The efficacy of enzyme replacement in the canine model of Hurlersyndrome using human IDUA generated in CHO cells was recently reported(Kakkis et al., 1995, Am. J. Hum. Genet. 57:A39 (Abstr.); Shull et al.,1994, supra). Weekly doses of approximately 1 mg administered over aperiod of 3 months resulted in normal levels of the enzyme in liver andspleen, lower but significant levels in kidney and Lungs and very lowlevels in brain, heart, cartilage and cornea (Shull et al., 1994, supra.Tissue examinations showed normalization of lysosomal storage in theliver, spleen and kidney, but no improvement in heart, brain and cornealtissues. One dog was maintained on treatment for 13 months and wasclearly more active with improvement in skeletal deformities, jointstiffness, corneal clouding and weight gain (Kakkis et al., 1995, supra.A single higher-dose experiment was quite promising and showeddetectable IDUA activity in the brain and cartilage in addition totissues which previously showed activity at the lower does. Additionalhigher-dose experiments and trials involving longer administration arecurrently limited by availability of recombinant enzyme. Theseexperiments underscore the potential of replacement therapy for Hurlerpatients and the severe constraints on both canine and human trials dueto limitations in recombinant enzyme production using currenttechnologies.

Lysosomal Enzymes: Biosynthesis and Targeting

Lysosomal enzymes are synthesized on membrane-bound polysomes in therough endoplasmic reticulum. Each protein is synthesized as a largerprecursor containing a hydrophobic amino terminal signal peptide. Thispeptide interacts with a signal recognition particle, an 11Sribonucleoprotein, and thereby initiates the vectoral transport of thenascent protein across the endoplasmic reticulum membrane into the lumen(Erickson, et al., 1981, J. Biol. Chem. 256:11224; Erickson, et al.,1983, Biochem. Biophys. Res. Commun. 115:275; Rosenfeld, et al., 1982,J. Cell Biol. 93:135). Lysosomal enzymes are cotranslationallyglycosylated by the en bloc transfer of a large preformedoligosaccharide, glucose-3, mannose-9, N-acetylglucosamine-2, from alipid-linked intermediate to the Asn residue of a consensus sequenceAsn—X—Ser/Thr in the nascent polypeptide (Kornfeld, R. & Kornfeld, S.,1985, Annu. Rev. Biochem. 54:631). In the endoplasmic reticulum, thesignal peptide is cleaved, and the processing of the Asn-linkedoligosaccharide begins by the excision of three glucose residues and onemannose from the oligosaccharide chain.

The proteins move via vesicular transport, to the Golgi stack where theyundergo a variety of posttranslational modifications, and are sorted forproper targeting to specific destinations: lysosomes, secretion, plasmamembrane. During movement through the Golgi, the oligosaccharide chainon secretory and membrane glycoproteins is processed to the sialicacid-containing complex-type. While some of the oligosaccharide chainson lysosomal enzymes undergo similar processing, most undergo adifferent series of modifications. The most important modification isthe acquisition of phosphomannosyl residues which serve as an essentialcomponent in the process of targeting these enzymes to the lysosome(Kaplan, et al., 1977, Proc. Natl. Acad. Sci. USA 74:2026). Thisrecognition marker is generated by the sequential action of two Golgienzymes. First, N-acetylglucosaminyl-phosphotransferase transfersN-acetylglucosamine-1-phosphate from the nucleotide sugar uridinediphosphate-N-acetylglucosamine to selected mannose residues onlysosomal enzymes to give rise to a phosphodiester intermediate (Reitman& Kornfeld, 1981, J. Biol. Chem. 256:4275; Waheed, et al., 1982, J.Biol. Chem. 257:12322). Then, N-acetylglucosamine-1-phosphodiesterα-N-acetylglucosaminidase removes N-acetylglucosamine residue to exposethe recognition signal, mannose-6-phosphate (Varki & Kornfeld, 1981, J.Biol. Chem. 256: 9937; Waheed, et al., 1981, J. Biol. Chem. 256:5717).

Following the generation of the phosphomannosyl residues, the lysosomalenzymes bind to mannose-6-phosphate (M-6-P) receptors in the Golgi. Inthis way the lysosomal enzymes remain intracellular and segregate fromthe proteins which are destined for secretion. The ligand-receptorcomplex then exits the Golgi via a coated vesicle and is delivered to aprelysosomal staging area where dissociation of the ligand occurs byacidification of the compartment (Gonzalez-Noriega, et al., 1980, J.Cell Biol. 85: 839). The receptor recycles back to the Golgi while thelysosomal enzymes are packaged into vesicles to form primary lysosomes.Approximately, 5-20% of the lysosomal enzymes do not traffic to thelysosomes and are secreted presumably, by default. A portion of thesesecreted enzymes may be recaptured by the M-6-P receptor found on thecell surface and be internalized and delivered to the lysosomes(Willingham, et al., 1981, Proc. Natl. Acad. Sci. USA 78:6967).

Two mannose-6-phosphate receptors have been identified. A 215 kDaglycoprotein has been purified from a variety of tissues (Sahagian, etal., 1981, Proc. Natl. Acad. Sci. USA, 78:4289; Steiner & Rome, 1982,Arch. Biochem. Biophys. 214:681). The binding of this receptor isdivalent cation independent. A second M-6-P receptor also has beenisolated which differs from the 215 kDa receptor in that it has arequirement for divalent cations. Therefore, this receptor is called thecation-dependent (M-6-P.sup.CD) while the 215 kDa one is calledcation-independent (M-6-P.sup.CI). The M-6-P.sup.CD receptor appears tobe an oligomer with three subunits with a subunit molecular weight of 46kDa.

Biosynthesis of Lysosomal Enzymes

Although many lysosomal enzymes are soluble and are transported tolysosomes by M-6-P receptors (MPR), integral membrane andmembrane-associated proteins such as human glucocerebrosidase (hGCB) aretargeted and transported to lysosomes independent of the M-6-P/MPRsystem (Kornfeld & Mellman, 1989, Erickson et al., 1985). hGCB does notbecome soluble after translation, but instead becomes associated withthe lysosomal membrane by means which have not been elucidated (vonFigura & Hasilik, 1986, Annu. Rev. Biochem. 55:167-193; Kornfeld andMellman, 1989, Annu. Rev. Cell Biol. 5:483-525). hGCB is synthesized asa single polypeptide (58 kDa) with a signal sequence (2 kDa) at theamino terminus. The signal sequence is co-translationally cleaved andthe enzyme is glycosylated with a heterogeneous group of both complexand high-mannose oligosaccharides to form a precursor. The glycans arepredominately involved in protein conformation. The “high mannose”precursor, which has a molecular weight of 63 KDa, ispost-translationally processed in the Golgi to a 66 KDa intermediate,which is then further modified in the lysosome to the mature enzymehaving a molecular weight of 59 KDa (Jonsson et al., 1987, Eur. J.Biochem. 164:171; Erickson et al., 1985, J. Biol. Chem., 260:14319).

The mature hGCB polypeptide is composed of 497 amino acids and containsfive N-glycosylation amino acid consensus sequences (Asn—X—Ser/Thr).Four of these sites are normally glycosylated. Glycosylation of thefirst site is essential for the production of active protein. Bothhigh-mannose and complex oligosaccharide chains have been identified(Berg-Fussman et al., 1993, J. Biol. Chem. 268:14861-14866). hGCB fromplacenta contains 7% carbohydrate, 20% of which is of the high-mannosetype (Grace & Grabowski, 1990, Biochem. Biophys. Res. Comm.168:771-777). Treatment of placental hGCB with neuraminidase (yieldingan asialo enzyme) results in increased clearance and uptake rates by ratliver cells with a concomitant increase in hepatic enzymatic activity(Furbish et al., 1981, Biochim. Biophys. Acta 673:425-434). Thisglycan-modified placental hGCB is currently used as a therapeutic agentin the treatment of Gaucher's disease. Biochemical and site-directedmutagenesis studies have provided an initial map of regions and residuesimportant to folding, activator interaction, and active site location(Grace et al., 1994, J. Biol. Chem. 269:2283-2291).

The complete complementary DNA (cDNA) sequence for hGCB has beenpublished (Tsuji et al., 1986, J. Biol. Chem. 261:50-53; Sorge et al.,1985, Proc. Natl. Acad. Sci. USA 82:7289-7293), and E. coli containingthe hGCB cDNA sequence cloned from fibroblast cells, as described (Sorgeet al., 1985, supra), is available from the American Type CultureCollection (ATCC) (Accession No. 65696).

Recombinant methodologies have the potential to provide a safer and lessexpensive source of lysosomal enzymes for replacement therapy. However,production of active enzymes, e.g., hGCB, in a heterologous systemrequires correct targeting to the ER, and appropriate N-linkedglycosylation at levels or efficiencies that avoid ER-based degradationor aggregation. Since mature lysosomal enzymes must be glycosylated tobe active, bacterial systems cannot be used. For example, hGCB expressedin E. coli is enzymatically inactive (Grace & Grabowski, 1990, supra).

Active monomers of hGCB have been purified from insect cells (Sf9 cells)and Chinese hamster ovary (CHO) cells infected or transfected,respectively, with hGCB cDNA (Grace & Grabowski, 1990, supra; Grabowskiet al., 1989, Enzyme 41:131-142). A method for producing recombinanthGCB in CHO cell cultures and in insect cell cultures was recentlydisclosed in U.S. Pat. No. 5,236,838. Recombinant hGCB produced in theseheterologous systems had an apparent molecular weight ranging from 64 to73 kDa and contained from 5 to 15% carbohydrate (Grace & Grabowski,1990, supra; Grace et al., 1990, J. Biol. Chem. 265:6827-6835). Theserecombinant hGCBs had kinetic properties identical to the natural enzymeisolated from human placentae, as based on analyses using a series ofsubstrate and transition state analogues, negatively-charged lipidactivators, protein activators (saposin C), and mechanism-based covalentinhibitors (Grace et al., 1994, supra; Berg-Fussman et al., 1993, supra;Grace et al., 1990, J. Biol. Chem. 265:6827-6835; Grabowski et al.,1989, supra). However, both insect cells and CHO cells retained most ofthe enzyme rather than secreting it into the medium, significantlyincreasing the difficulty and cost of harvesting the pure enzyme(Grabowski et al., 1989, supra). Accordingly, a recombinant system isneeded that can produce human or animal lysosomal enzymes in an activeform at lower cost, and that will be appropriately targeted for ease ofrecovery.

Enormous Costs of Pharmaceutical Enzyme Production

While the clinical treatment of Gaucher patients provides a dramaticallysuccessful example of an effective therapy, the expense underscores anequally inadequate production technology. For example, the present costfor the first year of treatment for a severely affected 70 kg patientwith Gaucher disease can reach $382,000. If the patient's clinicalparameters are not restored to normal in that time, treatment at thislevel of expense will be prolonged before dose reduction can beinitiated. Even with dose reduction, it is likely that the maintenancecost for such an individual will be in the range of $135,000 per year(at $3.70/IU). Many patients are unable to pay this large cost, andhealth carriers are extremely reluctant to underwrite this treatment forthe life of these patients. Cerezyme™ is as expensive as Ceredase™ andat this time is available only in limited quantities. The number ofpatients with Gaucher disease in the US currently receiving therapy isestimated to be only 10-15% of the total. According an article in NatureMedicine, since the introduction of this therapy six years ago the costof treating Gaucher patients worldwide will soon approach one billiondollars (19). Although the total number of patients worldwide who wouldbenefit from therapy is not known with any certainty, it is safe toassume that at least 80% of the world Gaucher population remainuntreated. To quote from the NIH Technology Assessment ConferenceSummary Statement, Feb. 27-Mar. 1, 1995. “As a prototype for all rarediseases, the plight of patients with Gaucher disease raises difficultfinancial and ethical issues, which we as a society must address (20).”Fabry disease is estimated to occur at a frequency of 1 in 40,000. Over400 hemizygous male patients have been clinically described. It isimperative that fundamentally new methods of enzyme production bedeveloped to reduce these costs so that all who suffer from these raredisorders can be treated.

Mammalian Lysosomes versus Plant Vacuoles

Because plants are eukaryotes, plant expression systems have advantagesover prokaryotic expression systems, particularly with respect tocorrect processing of eukaryotic gene products. However, unlike animalcells, plant cells do not possess lysosomes. Although the plant vacuoleappears functionally analogous to the lysosome, plants do not containMPRs (Chrispeels, 1991, Ann. Rev. Pl. Phys. Pl. Mol. Biol. 42:21-53;Chrispeels and Tague, 1991, Intl. Rev. Cytol. 125:1-45), and themechanisms of vacuolar targeting can differ significantly from those oflysosomal targeting. For example, the predominant mechanism of vacuolartargeting in plants does not appear to be glycan-dependent, but appearsto be based instead on C- or N-terminal peptide sequences (Gomez &Chrispeels, 1993, Plant Cell 5:1113-1124; Chrispeels & Raikhal, 1992,Cell 68:613-618; Holwerda et al., 1992, Plant Cell 4:307-318; Neuhaus etal., 1991, Proc. Natl. Acad. Sci. USA 88:10362-10366; Chrispeels, 1991,supra; Chrispeels & Tague, 1991, supra; Holwerda et al., 1990, PlantCell 2:1091-1106; Voelker et al., 1989, Plant Cell 1:95-104). As aresult, plants have not been viewed as appropriate expression systemsfor lysosomal enzymes which must be appropriately processed to producean active product.

An object of this invention is to provide the existing patientpopulation with enough active enzyme to develop a lower cost treatment.The enzymatic, structural, and glycan compositional analyses show rGalto be active. There are recent advances in glycoprotein modification anddrug delivery that allow, as examples, the chemical conjugation ofpeptides to carbohydrate, the covalent addition of polyethylene glycolto enzymes and the liposomal encapsulation of protein. Many additionalnew concepts can be tested to increase the half-life of enzymes incirculation and optimize cellular and subcellular targeting. Ideally,these modifications will require a facile and rapid genetic system toproduce large quantities of highly pure enzyme and an effective animaldisease model for drug development. Our lab-scale process appears highlyscalable and is capable of producing grams of enzyme per month inexisting indoor greenhouse growth areas.

Using a viral transfection system and transgenic plants, we haveexpressed enzymes in plants that have potential as therapeutic agentsfor humans with the metabolic storage disorders known as Fabry diseaseand Gaucher disease. High specific activity recombinant enzymes weresecreted by tobacco leaf cells via a default pathway of protein sortinginto the apoplastic compartment, a network of extracellular space, cellwall matrix materials and intercellular fluid (IF). We further developeda novel bioprocessing method to purify these enzymes from the IFfraction.

Another object of this invention is to provide an optimized preproenzymeamino acid (AA) sequence for secretion of highly active lysosomalenzymes. Another object of this invention is to provide an optimizedpurification of lysosomal enzymes from either the IF fraction or fromwhole plant homogenates. Another object of this invention is to providea molecular characterization of the enzymes purified by this process,including determination of enzyme specific activity.

SUMMARY OF THE INVENTION

The present invention provides for a polypeptide comprising (a) thecomplete, or a fragment of, the amino acid sequence of α-galactosidasewith or without (b) an ER-retention signal, such as the amino acidsequence SEKDEL (SEQ ID NO:37), wherein the ER-retention signal is atthe carboxy end of the complete, or a fragment of, the amino acidsequence of α-galactosidase, wherein said fragment of the amino acidsequence of α-galactosidase comprises a deletion of at the carboxy endof α-galactosidase, wherein said deletion is one to twenty-five aminoacids. The present invention also provides for a polynucleotide encodingthe aforementioned polypeptide.

The present invention also relates to the production of these human oranimal lysosomal enzymes, including the aforementioned polypeptides, intransformed or transfected plants, plant cells or plant tissues, andinvolves constructing and expressing recombinant expression constructscomprising lysosomal enzyme coding sequences in a plant expressionsystem. The plant expression system provides appropriateco-translational and post-translational modifications of the nascentpeptide required for processing, e.g., signal sequence cleavage,glycosylation, and sorting of the expression product so that anenzymatically active protein is produced. Using the methods describedherein, recombinant lysosomal enzymes are produced in plant expressionsystems from which the recombinant lysosomal enzymes can be isolated andused for a variety of purposes.

The present invention is exemplified by virally transfected andtransgenic tobacco plants with lysosomal enzyme expression constructs.One construct comprises a nucleotide sequence encoding a modified humanglucocerebrosidase (hGCB). Another construct comprises nucleotidesequence encoding a human α galactosidase (α gal or α gal A). Virallytransfected and transgenic tobacco plants having the expressionconstructs produce lysosomal enzymes that are enzymatically active andhave high specific activity.

The plant expression systems and the recombinant lysosomal enzymesproduced therewith have a variety of uses, including but not limited to:(1) the production of enzymatically active lysosomal enzymes for thetreatment of lysosomal storage diseases; (2) the production of alteredor mutated proteins, enzymatically active or otherwise, to serve asprecursors or substrates for further in vivo or in vitro processing to aspecialized industrial form for research or therapeutic uses, such as toproduce a more effective therapeutic enzyme; (3) the production ofantibodies against lysosomal enzymes for medical diagnostic use; and (4)use in any commercial process that involves substrate hydrolysis.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows a Tobamovirus expression vectors.

FIG. 2 shows a Tobamovirus expression vector containing the human αgalactosidase gene or a variant of the gene. The amino acid sequence ofαASP and Native SP depicted in FIG. 2 are depicted in SEQ ID. NO: 1 and2, respectively. The entire amino acid sequence of WT rGAL-A is depictedin SEQ ID NO: 4.

FIG. 3A shows accumulation by Western Analysis of total plant solubleextract anti human GAL-A sera.

FIG. 3B shows activity of WT rGAL-A at 8 and 14+ days post inoculationof the plant host with a viral vector.

FIG. 4A shows Western blot analysis of total plant soluble extract antihuman GAL-A sera

FIG. 4B shows activity of WT rGAL-wt and rGAL-wtR at 8 and 14+ days postinoculation of the plant host with a viral vector.

FIG. 5 shows carboxy terminal modifications to α galactosidase. Theasterisk indicates a potential CTPP cleavage site according to Gene 58:177, 1987. The entire nucleic acid and amino acid sequences of WTrGAL-A, WT-rGAL-AR, rGAL-4, rGAL-4R, rGAL-8, rGAL-8R, rGAL-12, rGAL-12R,rGAL-25, and rGAL-25R are depicted in SEQ ID NO: 3-22, respectively.

FIG. 6 shows western blot analysis of the accumulation of 10carboxy-modified rGAL-A variants from interstitial fluid and from totalplant homogenate.

FIG. 7 shows a comparison of enzymatic activity of the 10carboxy-modified rGAL-A variants.

FIG. 8 shows a Coomassie blue stained electrophoresis gel separation ofcarboxy-modified rGAL-A variants and controls.

FIG. 9 shows a Coomassie blue stained electrophoresis gel separation ofcarboxy-modified rGAL-A variants and controls.

FIG. 10 shows a schematic representation of rGAL-A secretion from theendoplasmic reticulum to the apoplast.

FIG. 11 shows different glycosylation structures of α galactosidase.

FIG. 12 shows TTODA (rGAL-12R) TMV RNA begins at base 1; 126/183 readingframe begins at 69, 3417 is suppressible stop codon, and ends at4919.30K ORF begins at 4903 and ends at 5709. Human α galactosidase ARNA begins at 5703, α amylase signal peptide is from 5762-5857; maturehuman α galactosidase A coding region is 5858-7036, ToMV virus coatprotein and 3 UTR follows. (SEQ ID NO: 33)

FIG. 13 shows SBS5-rGAL-12R TMV RNA begins at base 1; 126/183 readingframe begins at 69, 3417 is suppressible stop codon, and ends at4919.30K ORF begins at 4903 and ends at 5709. Human α galactosidase ARNA begins at 5703, complete (signal peptide and mature protein codingregion) human α galactosidase A gene 5766-7037, TMV U5 virus coatprotein and 3 UTR follows (SEQ ID. NO: 34).

FIG. 14 shows the construct within pBSG638: a dual Cauliflower MosiacVirus 35S promoter linked to a translational enhancer from Tobacco EtchVirus linked 5′ to, and a polyadenylation region from the noplinesynthase gene of Agrobacterium tumefaciens linked 3′ to, the nativehuman GCB cDNA.

FIG. 15 shows the construct within pBSG641: a dual Cauliflower MosiacVirus 35S promoter linked 5′ to, and a polyadenylation region from thenopline synthase gene of Agrobacterium tumefaciens linked 3′ to, theentire genome of Tobacco Mosaic Virus except the coat protein region isreplaced with the GCB gene.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a polynucleotide comprising thenucleotide sequence depicted in SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17,19, 31, or 32. The present invention also provides a polypeptidecomprising the amino acid sequence depicted in SEQ ID NO: 4, 6, 8, 10,12, 14,16, 18, or 20.

The present invention further provides for a polypeptide comprising (a)the complete, or a fragment of, the amino acid sequence ofα-galactosidase and (b) the amino acid SEKEL (SEQ ID NO:37), whereinSEKEL is at the carboxy end of the complete, or a fragment of, the aminoacid sequence of α-galactosidase, wherein said fragment of the aminoacid sequence of α-galactosidase comprises a deletion of at the carbozyend of α-galactosidase, wherein said deletion is one to twenty-fiveamino acids. Preferably, the deletion is one to twelve amino acids. Morepreferably, the deletion is four to twelve amino acids. The presentinvention further provides for a polynucleotide comprising a nucleotidesequence encoding the aforementioned polypeptide(s).

The present invention further provides for a polynucleotide encoding theaforementioned polypeptide(s).

The present invention also provides for a viral vector or expressionvector comprising the aforementioned polynucleotide(s) or encoding theaforementioned polypeptide(s). The viral vector or expression vector iscapable of expression and systemic expression of the polypeptide(s)encoded by the polynucleotide in a plant cell or a plant. Preferably,the viral vector or expression vector is derived from or based on orobtained from an RNA virus or an RNA viral vector. More preferably, theRNA virus is an RNA plant virus. Even more preferably, the RNA plantvirus is a single-stranded plus-sense RNA plant virus. The RNA plantvirus is multi-partite, monopartite, bipartite, tripartite, or the like.Even much more preferably, the single-stranded plus-sense RNA plantvirus is a tobamovrius, such as a tobacco mosaic virus.

The present invention further provides for a plant cell or a plantexpressing the aforementioned polypeptide(s).

The present invention provides for a method for producing a protein ofchoice comprising a lysosomal enzyme which is enzymatically active,comprising: recovering the lysosomal enzyme from (i) a transgenic plantcell or (ii) a cell, tissue or organ of a transgenic plant, whichtransgenic plant cell or plant is transformed or transfected with arecombinant expression construct comprising a nucleotide sequenceencoding the lysosomal enzyme and a promoter that regulates expressionof the nucleotide sequence so that the lysosomal enzyme is expressed bythe transgenic plant cell or plant.

The promoter can be an inducible promoter. The inducible promoter can beinduced by mechanical gene activation. The method can be carried outwith the transgenic plant and additionally comprises a step of inducingthe inducible promoter before or after the transgenic plant isharvested, which inducing step is carried out before recovering thelysosomal enzyme from the cell, tissue or organ of the transgenic plant.

The lysosomal enzyme can be a modified lysosomal enzyme which isenzymatically active and comprises: (a) an enzymatically-active fragmentof a human or animal lysosomal enzyme; (b) the human or animal lysosomalenzyme or (a) having one or more amino acid residues added to the aminoor carboxyl terminus of the human or animal lysosomal enzyme or (a); or(c) the human or animal lysosomal enzyme or (a) having one or morenaturally-occurring amino acid additions, deletions or substitutions.The modified lysosomal enzyme can comprise a signal peptide ordetectable marker peptide at the amino or carboxyl terminal of themodified lysosomal enzyme. The modified lysosomal enzyme can berecovered from (i) the transgenic plant cell or (ii) the cell, tissue ororgan of the transgenic plant by reacting with an antibody that bindsthe detectable marker peptide. The antibody can be a monoclonalantibody.

The modified lysosomal enzyme can comprise: (a) an enzymatically-activefragment of an α-N-acetylgalactosaminidise, acid lipase,α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronaksulfatase, α-mannosidase or sialidase; (b) theα-N-α-cetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidase,sialidase or (a) having one or more amino acid residues added to theamino or carboxyl terminus of the α-N-acetylgalactosaminidase, acidlipase, α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase, sialidase or (a); or (c) theα-N-acetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidase,sialidasc or (a) having one or more naturally-occurring amino acidadditions, deletions or substitutions.

The modified lysosomal enzyme can comprise: (a) an enzymatically-activefragment of a human glucocerebrosidase or human α-L-iduronidase enzyme;(b) the human glucocerebrosidase, human α-L-iduronidase or (a) havingone or more amino acid residues added to the amino or carboxyl terminusof the human glucocerebrosidase, human α-L-iduronidase or (a); or (c)the human glucocerebrosidase, human α-Liduronidase or (a) having one ormore naturally-occurring amino acid additions, deletions orsubstitutions.

The modified lysosomal enzyme can be a fusion protein comprising: (I)(a) the enzymatically-active fragment of the human or animal lysosomalenzyme, (b) the human or animal lysosomal enzyme, or (c) the human oranimal lysosomal enzyme or (a) having one or more naturally-occurringamino acid additions, deletions or substitutions, and (II) a cleavablelinker fused to the amino or carboxyl terminus of (I); and the methodcomprises: (a) recovering the fusion protein from the transgenic plantcell, or the cell, tissue or organ of the transgenic plant; (b) treatingthe fusion protein with a substance that cleaves the cleavable linker sothat (1) is separated from the cleavable linker and any sequenceattached thereto; and (c) recovering the separated (I).

The transgenic plant can be a transgenic tobacco plant. The lysosomalenzyme can be a human or animal lysosomal enzyme. The lysosomal enzymecan be an α-N-cetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseor sialidese. The lysosomal enzyme can be a human glucocerebrosidase orhuman α-L-iduronidase. The organ can be a leaf, stem, root, flower,fruit or seed.

The present invention provides for a recombinant expression constructcomprising a nucleotide sequence encoding a protein of choice comprisinga lysosomal enzyme and a promoter that regulates the expression of thenucleotide sequence in a plant cell.

The promoter can be an inducible promoter. The inducible promoter can beinduced by mechanical gene activation. The lysosomal enzyme can be amodified lysosomal enzyme which is enzymatically active and comprises:(a) an enzymatically active fragment of a human or animal lysosomalenzyme; (b) the human or animal lysosomal enzyme or (a) having one ormore amino acid residues added to the amino or carboxyl terminus of thehuman or animal lysosomal enzyme or (a); or (c) the human or animallysosomal enzyme or (a) having one or more naturally-occurring aminoacid additions, deletions or substitutions. The modified lysosomalenzyme can comprise a signal peptide or detectable marker peptide at theamino or carboxyl terminal of the modified lysosomal enzyme.

The modified lysosomal enzyme can comprise (a) an enzymatically-activefragment of an α-N-acetylgalactosaminidase, acid lipase,α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase or sialidase; (b) theα-N-acetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidase,sialidase or (a) having one or more amino acid residues added to theamino or carboxyl terminus of the α-N-acetylgalactosaminidase, acidlipase, α-galactosidase, glucocerebrosidase, cr-Liduronidase, iduronatesulfatase, cr-mannosidase, sialidase or (a); or (c) theα-N-acetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidase,sialidase or (a) having one or more naturally-occurring amino acidadditions, deletions or substitutions.

The modified lysosomal enzyme can comprise (a) an enzymatically-activefragment of a human glucocerebrosidase or human α-L-iduronidase enzyme;(b) the human glucocerebrosidase or human α-L-iduronidase or (a) havingone or more amino acid residues added to the amino or carboxyl terminusof the human glucocerebrosidase, human α-L-iduronidase or (a); or (c)the human glucocerebrosidase, human (α-L-iduronidase or (a) having oneor more naturally-occurring amino acid additions, deletions orsubstitutions.

The modified lysosomal enzyme can be a fusion protein comprising: (I)(a) the enzymatically-active fragment of the human or animal lysosomalenzyme, (b) the human or animal lysosomal enzyme, or (c) the human oranimal lysosomal enzyme or (a) having one or more naturally-occurringamino acid additions, deletions or substitutions, and (II) a cleavablelinker fused to the amino or carboxyl terminus of (I). The lysosomalenzyme can be a human or animal lysosomal enzyme. The lysosomal enzymecan be an α-N-acetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseor sialidase. The lysosomal enzyme can be a human glucocerebrosidase orhuman α-L-iduronidase.

The present invention provides for a plant transformation ortransfection vector comprising any of the recombinant expressionconstruct recited above.

The present invention provides for a plant which is transformed ortransfected with any of the recombinant expression construct recitedabove.

The present invention provides for a plant cell, tissue or organ whichis transformed or transfected with any of the recombinant expressionconstruct recited above.

The present invention provides for a plasmid comprising any of therecombinant expression construct recited above.

The present invention provides for a transgenic plant or plant cellcapable of producing a lysosomal enzyme which is enzymatically active,which transgenic plant or plant cell is transformed or transfected witha recombinant expression construct comprising a nucleotide sequenceencoding a lysosomal enzyme and a promoter that regulates expression ofthe nucleotide sequence in the transgenic plant or plant cell. Thepromoter is an inducible promoter. The inducible promoter is induced bymechanical gene activation. The lysosomal enzyme which is a modifiedlysosomal enzyme which is enzymatically active and which comprises: (a)an enzymatically-active fragment of a human or animal lysosomal enzyme;(b) the human or animal lysosomal enzyme or (a) having one or more aminoacid residues added to the amino or carboxyl terminus of the human oranimal lysosomal enzyme or (a); or (c) the human or animal lysosomalenzyme or (a) having one or more naturally-occurring amino acidadditions, deletions or substitutions. The modified lysosomal enzymecomprises a signal peptide or detectable marker peptide at the amino orcarboxyl terminal of the modified lysosomal enzyme.

The modified lysosomal enzyme comprises: (a) an enzymatically-activefragment of an α-N-acetylgalactosaminidase, acid lipase,α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase or sialidase; (b) theα-N-acetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidase,sialidase or (a) having one or more amino acid residues added to theamino or carboxyl terminus of the α-N-acetylgalactosaminidase, acidlipase, α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, amannosidase, sialidase or (a); or (c) theα-N-acetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidase,sialidase or (a) having one or more naturally-occurring amino acidadditions, deletions or substitutions.

The modified lysosomal enzyme can comprise: (a) an enzymatically-activefragment of a human glucocerebrosidase or human α-L-iduronidase enzyme;(b) the human glucocerebrosidase, human α-L-iduronidase or (a) havingone or more amino acid residues added to the amino or carboxyl terminusof the human glucocerebrosidase, human α-L-iduronidase or (a); or (c)the human glucocerebrosidase, human α-L-iduronidase or (a) having one ormore naturally-occurring amino acid additions, deletions orsubstitutions.

The modified lysosomal enzyme is a fusion protein comprising: (I) (a)the enzymatically-active fragment of the human or animal lysosomalenzyme, (b) the human or animal lysosomal enzyme, or (c) the human oranimal lysosomal enzyme or (a) having one or more naturally-occurringamino acid additions, deletions or substitutions, and (II) a cleavablelinker fused to the amino or carboxyl terminus of (I). The transgenicplant or plant cell is a transgenic tobacco plant or tobacco cell. Thelysosomal enzyme is a human or animal lysosomal enzyme. The lysosomalenzyme is an α-N-acetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseor sialidase. The lysosomal enzyme is a human glucocerebrosidase orhuman α-L-iduronidase.

The present invention provides for a leaf, stem, root, flower or seed ofany of the transgenic plant recited above.

The present invention provides for a seed of plant line Nicotiana sp.,which seed has the ATCC Accession No. PTA-2258, deposited Jul. 25, 2000.

The present invention provides for a plant grown from the seed recitedabove.

The present invention provides for a lysosomal enzyme which isenzymatically active and is produced according to a process comprising:recovering the lysosomal enzyme from (i) a transgenic plant cell or (ii)a cell, tissue or organ of a transgenic plant which transgenic plantcell or plant is transformed or transfected with a recombinantexpression construct comprising a nucleotide sequence encoding thelysosomal enzyme and a promoter that regulates expression of thenucleotide sequence so that the lysosomal enzyme is expressed by thetransgenic plant cell or plant. The promoter can be an induciblepromoter. The process is carried out with the transgenic plant andadditionally can comprise a step of inducing the inducible promoterbefore or after the transgenic plant is harvested, which inducing stepis carried out before recovering the lysosomal enzyme from the cell,tissue or organ of the transgenic plant.

The modified lysosomal enzyme which can be enzymatically active and cancomprise: (a) an enzymatically-active fragment of a human or animallysosomal enzyme; (b) the human or animal lysosomal enzyme or (a) havingone or more amino acid residues added to the amino or carboxyl terminusof the human or animal lysosomal enzyme or (a); or (c) the human oranimal lysosomal enzyme or (a) having one or more naturally-occurringamino acid, additions, deletions or substitutions. The modifiedlysosomal enzyme can comprise a signal peptide or detectable markerpeptide at the amino or carboxyl terminal of the modified lysosomalenzyme.

The modified lysosomal enzyme can comprise: (a) an enzymatically-activefragment of an α-N-acetylgalactosaminidase, acid lipase,α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, α-mannosidase or sialidase; (b) theα-N-acetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-Liduronidase, iduronate sulfatase, α-mannosidase,sialidase or (a) having one or more amino acid residues added to theamino or carboxyl terminus of the α-N-acetylgalactosatninidase, acidlipase, α-galactosidase, glucocerebrosidase, α-L-iduronidase, iduronatesulfatase, amannosidase, sialidase or (a); or (c) theα-N-acetylgalactosaminidasd, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfazase, α-mannosidase,sialidase or (a) having one or more naturally-occurring amino acidadditions, deletions or substitutions.

The modified lysosomal enzyme comprises: (a) an enzymatically-activefragment of a human glucocerebrosidase or human α-L-iduronidase enzyme;(b) the human glucocerebrosidase, human α-L-iduronidase or (a) havingone or more amino acid residues added to the amino or carboxyl terminusof the human glucocerebrosidase, human α-L-iduronidase or (a); or (c)the human glucocerebrosidase, human α-L-iduronidase or (a) having one ormore naturally-occurring amino acid additions, deletions orsubstitutions. The modified lysosomal enzyme can be a fusion proteincomprising: (I) (a) the enzymatically-active fragment of the human oranimal lysosomal enzyme, (b) the human or animal lysosomal enzyme, or(c) the human or animal lysosomal enzyme or (a) having one or morenaturally-occurring amino acid additions, deletions or substitutions,and (II) a cleavable linker fused to the amino or carboxyl terminus of(I).

The transgenic plant can be a transgenic tobacco plant. The lysosomalenzyme can be a human or animal lysosomal enzyme. The lysosomal enzymecan be an α-N-acetylgalactosaminidase, acid lipase, α-galactosidase,glucocerebrosidase, α-L-iduronidase, iduronate sulfatase, α-mannosidaseor sialidase. The lysosomal enzyme can be a human glucocerebrosidase orhuman α-L-iduronidase. The organ can be a leaf, stem, root, flower,fruit or seed.

Gal-A is one of many proteins that require glycan site occupancy atN-linked sites to achieve proper folding and stability. The ability tosuccessfully target the enzyme in Fabry patients is also likely to beglycosylation-dependent. This requirement presently limits theexpression possibilities to eukaryotic cell types. Recombinant proteinssynthesized in baculovirus and yeast expression systems are oftenhyperglycosylated and highly heterogeneous complicating the preparationof therapeutically effective glycoforms from these sources. The rGal-Asynthesized in plants is a relatively homogeneous glycoform as analyzedby its SDS-PAGE electrophoretic mobility and comigrates with rGal-Aproduced purified from placenta (FIG. 3). The expression results (yieldand purity) we have already presented are unprecedented in any eukaryotesystem for a glycosylated enzyme and are not likely to be achieved inthe foreseeable future with transgenic plants or animals. “Crude” rGal-Afrom the leaf IF has a specific activity of over 1,000,000 U/mg ofprotein, whereas CHO, COS-1 and insect cell extracts and supernatantsare maximally only 10-20,000 U/mg; (36,41,42).

Protein pharmaceuticals may vary over five orders of magnitude in unitvalue and be required in kg/year quantities. The example of Gaucherdisease emphasizes the need progress in production phase research. Manyadditional heritable metabolic disorders, particularly those caused bydysfunctional lysosomal enzymes, might be treated by supplementationwith exogenously produced enzymes. Enzyme replacement usingmacrophage-targeted human glucocerebrosidase has been shown to beextraordinarily beneficial for Gaucher patients. However, the cost ofthis treatment is very great. If the significant advances in clinicalresearch are to be applied on a practical scale, new productiontechnologies will be required to deliver bioproducts such as these tothose in need at an affordable cost (43). No savings in Gauchertreatment costs were realized upon introduction of the recombinantCHO-cell product Cerezyme™ to replace the placental-derived Ceredase™. Asignificant reduction in cost requires fundamental changes in both thesource of enzyme and process of purification.

While the existing treatment for Gaucher disease is safe and effective,there are potential contaminants derived from the source material thatmay pose serious risks. For the existing pharmaceutical products, theserisks primarily include possibilities of contamination with humanpathogenic viruses or peptides with potent hormonal activities such ashuman chorionic gonadotropin (44). These potential contaminants are notpresent in plant source material.

The main goal in selecting plants for expression of this protein is thepotential for a radical reduction in costs. For the RNA-viral mediatedsynthesis of rGal-A and rGCB in plants, this is very likely to beachieved through the synergistic combination of three factors:

Complex crude extracts from various eukaryote cell production systemsmay be replaced with a plant leaf homogenate or IF fractions highlyenriched in recombinant product.

Large-scale, sterile, cell fermentation systems and associated media,capitalization, and waste costs may be replaced with plant biomass.Production is then inexpensively scaled to the quantities desired.

The labor and time required to generate transgenic higher plants oranimals may be replaced with a very rapid and simple planttransformation or plant viral transfection system.

Modern agriculture can supply a new generation of medicinal plants as asource of pharmaceuticals—a source that should be as inexpensive andreadily available as our food, fiber, flavors and chemical feedstocks.

Experimental Design and Methods

Post-Translational Processing and Secretion. Protein secretion to theplant apoplast is through a default pathway. In our experience, additionof the rice α-amylase signal peptide (α-ASP) sequence at the N-terminusof several recombinant proteins is sufficient to direct the protein tothe lumen of the ER in tobacco leaves. However, this is not likely to bea rate limiting step in protein accumulation and many native signalpeptides may function equally well in plants. It would be most desirableto include few if any additional AA residues at the N terminus afterprocessing. For this reason, we compared expression from the nativesignal peptide encoded in human Gal-A cDNA to that from the foreignα-ASP sequence.

Mutations in the carboxy-terminal domain of the Gal-A homodimer haveprofound effects on enzymatic activity. Several mutations occurring inindividuals affected with Fabry disease map to this region. Some ofthese mutations have a dominant negative phenotype. When a peptide mapwas published on Gal-A purified from human lung, the authors noted theabsence of the most carboxy-terminal predicted fragment and hypothesizedthe proteolytic removal of 26 or 28 AA from this region (39,40). Veryrecently Miyamura et al. published a study of the effects ofcarboxy-terminal truncations on enzymatic activity in transfected COS-1cells (42). Between 2 and 17 AA residues were removed by introducingstop codons into a series of cDNA constructs. Relative enzyme activity,measured using 4-MUG as a substrate, first increased and then decreasedas AA were removed. 12 and 17 AA constructs had no activity, while 11was the same as wild type. A 4 AA construct yielded the highest activityat approximately 6.2× the full-length sequence. Because the precise AAsequence of the carboxy-terminus of the native human enzyme was neverdetermined, there is insufficient information to interpret theseresults. The carboxy-terminal domain may affect the conformation of theactive site either directly or indirectly through a proteolyticmaturation step and/or assembly and subcellular localization of theactive form. Furthermore, it is important to stress that it is theenzyme activity on galactose-terminal glycosphingolipids that isrelevant to development of a therapeutic enzyme.

Plant proteins do not require N-linked oligosaccharides for correctsorting into vacuoles (35, 37, 38). Some vacuolar proteins (osmotin,thaumatin, chitinase-I, glucanase-I and a barley lectin), containsorting information in a CTPP of 7 to 22 AA in length. For several ofthese proteins secreted isoforms are synthesized without a CTPP domain.In other cases, experimental deletion of the CTPP results in secretionof the recombinant protein to the IF (45-48). Sorting of Gal-A to thelysosome is likely to occur by the well-characterizedmannose-6-phosphate receptor pathway in mammalian cells. We hypothesizethat a redundant sorting signal may exist in this carboxy-domain thatalso serves to reduce enzymatic activity in the ER lumen, golgi andtrans-golgi network. This signal appears to function in plant cells,presumably for vacuolar localization.

Scale-up Purification and Analysis. In order to evaluate the performanceof larger scale process equipment, we designed and had built a custombasket centrifuge fixture for a laboratory low-speed centrifuge that hasa capacity of approximately 1 kilogram leaf material. The sensitivity ofthe fluorescent 4-MUG enzyme assay allowed us to begin to evaluateenzyme purification from the leaf IF fraction using the constructrGal-A-SEKDEL. (This vector only yields approximately {fraction(1/50)}th the activity of rGal-A12-SEKDEL). Leaf tissue was transfected,harvested and infiltrated as described in Section B4 (ExperimentalResults). Galactosidase activity was stable in crude IF extracts and wasbound to the hydrophobic interaction resin octyl sepharose, and elutedin a descending ammonium sulfate gradient. The lectin resinconcanavalin-A sepharose was also effective, indicating the presence ofat least one high mannose chain. The enzyme did not bind to acommercially available melibiose column (Sigma).

We have measured the enrichment provided by the affinity resinα-galactosylamine Sepharose with a C12 arm (49). Some or all of thethree effective chromatography steps were combined as necessary with asize exclusion fractionation to yield highly purified enzyme(s). Becauseour current source of enzyme is so enriched (FIG. 3), and several of thepublished purification steps we have shown to be compatible with theplant IF extracts, we anticipate no problems in enzyme purification.Pure enzyme preparations were shipped to the laboratory of Drs. RoscoeBrady and Gary Murray for evaluation of enzyme activity with¹⁴C-galactose-labeled ceramide trihexoside. These colleagues wereresponsible for the development of the therapeutically effectiveglycoform of glucocerebrosidase used to treat Gaucher disease.

We scaled up the purification of up to four candidate therapeuticenzymes as necessary in our indoor greenhouses. In our initialexperiment, 38 and 48 percent of the total rGal-A activity was recoveredupon the first infiltration and centrifugation treatment (ConstructrGal-A12-SEKDEL) for a yield of >50 mg of enzyme per kilogram of leafmaterial. Experience with the extraction of glucocerebrosidase from theIF indicates that additional enzyme is recovered in a second treatment.In these experiments one leaf was collected for each sample from each oftwo plants. There was considerable plant to plant variation in the levelof enzyme activity. We analyzed more carefully the accumulation ofenzyme activity over time post-inoculation to optimize yields. Ourfacilities are more than sufficient to provide the 1 kilogram quantitiesof biomass necessary to purify nanomoles of enzyme for the followingsequence and structural work. Sequence analysis and MALDI-TOF molecularweight determination was performed as a commercial service byCommonwealth Biotechnologies, Inc. N-terminal sequence is by theautomated Edman degradation. C-terminal sequence is by carboxypeptidasedigestion followed with amino acid analysis.

Full-scale Bioprocess Pilot Plant

Macroextraction. Large-scale maceration of tissue was accomplished by a65 hp Rietz disintegrator mill. The macerated tissue was then separatedinto a “green juice” fraction and a fiber fraction in a Rietz screwpress. The fiber fraction was dried in a Cardwell drier. The “greenjuice” was then pH adjusted and heated in a dual plate-and-frame heatexchanger system with adjustable holding tube. The process of pHadjustment and heating causes the precipitation of the F1 proteincomplex. The protein was then clarified in a 40 hp. Westphalia SA-40disk stack centrifuge capable of clarification of “green juice” atgreater than 20 gallons per minute (GPM).

Downstream Processing. The concentrate was then pumped to Clean Room 1that houses the primary ultrafiltration (UF) equipment. This equipmentwas fitted with over 1000 sq. ft. of spiral wound membranes. Typically,the UF was equipped with 100,000 kDa cut-off membranes. Virus particlesare recovered in the retentate. Lower molecular weight proteins arerecovered in the permeate. The permeate was fractionated by a second UFsystem fitted with appropriate molecular weight cut-off membranes. Theretentate was processed in Clean Room 2. Virus was recovered bypolyethylene glycol (PEG) precipitation and centrifugation in twoSharples vertical bowl centrifuges. Final purification of solubleproteins and peptides was accomplished on a series of chromatographysystems.

Additional Facilities. The facility has other major unit processesavailable for the recovery and purification of plant fractions. Thereare two Alar diatomaceous earth, rotary vacuum filters. One of thefilters was in an explosion proof area of the pilot plant that can beused for solvent extraction. The solvent extraction facility also has abiphasic solvent extractor and high efficiency distillation column.Extensive tankage was available both indoors and outdoors. Pumps,filters and other process equipment are available at the facility,allowing a large margin of flexibility while developing new processes.

Full-Scale Pilot Plant Implementation. The Bioprocess Facility hasexcellent supporting infrastructure. The 900 square foot laboratory wasequipped with all the basic tools for biochemical and protein analysesincluding: electrophoresis, gel filtration, HPLC, spectrophotometry,basic chromatography, chemical analysis, and sample preparation andpreservation. The full scale pilot plant has approximately 15,000 squarefeet of additional floor space for expansion including a high bay tower.External solvent tanks are placed in diked enclosures. Two rapidrecovery, high pressure (up to 600 psi) steam generators and a largetwin screw, oilless compressor are on site. A complete shop andmaintenance facility was present along with walk-in cold room andwalk-in freezer. Additional equipment includes a ceramic microfiltrationsystem, a spray dryer and an array of tanks, pumps, filters, heatexchangers, and agitators.

Process equipment was fabricated and modified by a group of skilledvendors and craftsmen capable of fabricating specialized equipmentdesigned by the company, and has excellent field experience working inlarge scale operations.

Infiltration System. Vacuum infiltration can be accomplished in thefield or at the processing facility. Development experiments determinethe necessity to infiltrate the material in the field. A vacuum tank wasused as the receiver for the plant tissue after harvest by the tobaccocutter. The tissue was conveyed into a trailer-mounted tank capable offull vacuum and slurried into an infiltration buffer. The Owensborofacility has a trailer capable of carrying approximately 18,000 lb. Thiswill translated into approximately 1,000 gallons per trip to the field.The trailer was fitted with a 2,000 gallon tank capable of full volumeand evacuated by a gasoline driven vacuum pump. In harvests from1991-1994, it was the goal of the team to have harvested biomass at theprocessing facility in less than 1 hour after cutting. If the tissue canbe held for approximately one hour without significant loss of enzymeactivity, the biomass can be brought from the field in the conventionalwagon and infiltrated at the processing facility. Several large, fullvacuum tanks can be employed at the facility to increase the totalthroughput of the plant. Two large-scale vacuum pump systems in theplant that are currently associated with the Alar rotary vacuum filterscan be used for the vacuum infiltration process step.

Basket Centrifugation. The full-scale basket-type centrifuge was adiscontinuous batch-type system. Leaf tissue can be slurried in,dewatered as a batch, then a scraper system discharges the solids to abottom dump. Large leaves and pieces of tissue can be handled in thismanner. The potential of placing a vacuum system on the discharge sideof the centrifuge was also be investigated. The centrifuge was ahydraulically driven conventional basket centrifuge with a bottomdischarge and bowl dimension of 48 inches in diameter and a depth of 30inches. Optimum loadings of the centrifuge in full-scale was determinedthe throughput and cycle times of this process step.

Vacuum Extraction. Vacuum extraction can be accomplished in large-scaleby a web or belt-type vacuum filter system common in the food ingredientbusiness. The “in-plant” vacuum systems could also be adapted to operatethis type of filter. The plant tissue can be placed on this type offilter before or after the centrifugation step. Some damage of thebiomass was anticipated during the scraper mediated discharge of thebasket centrifuge. The discharged material was analyzed for the presenceof intracellular components and their effect on enzyme activity,recovery and separation. These data determined the position of thevacuum filtration step in the process flow.

Downstream Processing. The initial UF separation was accomplished by anAlfa-Laval custom UF system consisting of six modular housings eachcontaining either three 12 inch spiral wound membranes (Amicon type) orone standard 38 inch module. This yields a UF system with between 740and 1140 square feet of membrane area of typical spiral woundconfiguration. The ability to interchange housings and replace housingsby spool pieces gives the system great flexibility in large-scaleprocess development. This system was housed in Clean Room #1. This roomis 14×18 ft, and is under positive pressure, HEPA-filtered air. A secondUF system was available in Clean Room #1. This smaller system, built bySeparations Equipment Technology (SETEC), has the capacity for 320square feet of spiral wound membrane. This system was employed for thesecond separation and diafiltration. It was designed for automaticdiafiltration. Clean Room #2 is equipped with a Pharmacia Streamlinefluid bed gel filtration system equipped with UV and refractive indexmonitoring equipment. This unit was available for chromatography steps.

An antiserum specific for these xylose- and fucose-containing complexglycans was especially useful in developing an ELISA assay to followenzymatic deglycosylation. Large quantities of purified enzymefacilitate definitive determination of glycosylation structure and ifnecessary provide adequate rGal-A to use as substrate for enzymaticdeglycosylation reactions. Using Gal-A knockout mice in the laboratoryof Dr. Brady at NIH was an important genetic tool in developing atherapeutically effective glycoform. We use our transfected plants as aconvenient source of recombinant enzyme for glycan analysis. Glycoformsare shown in FIG. 11.

Plants as a Source of Recombinant Pharmaceutical Proteins. A number ofgenetic tools have been developed during the last decade for theexpression of foreign genes in plants. In addition to various antibodymolecules (21-23), the accumulation of serum proteins (24) and candidatevaccine products (25-28) has been described in the leaves and othertissues of whole plants. We increased the attainable expression levelsthrough the use of chimeric RNA viruses. For production of specificproteins, transient expression of foreign genes in plants usingvirus-based vectors has several advantages. These chimeric viruses movequickly from an initial infection site and deliver the recombinant geneto essentially all somatic cells of the plant. The gene vectors arepremier analytical tools because they allow both high level expressionand brief cycles of protein modification and testing. A permissive hostprovides high levels of expression and may be used for rapid,large-scale recombinant protein production in whole plants.

We validated the performance of plant-based expression systems for theproduction of recombinant proteins and peptides of pharmaceuticalsignificance. In two weeks post-inoculation, the ribosome inactivatingenzyme α-trichosanthin was over-produced in plants to 2% of the totalsoluble protein and had the same specific activity as the enzyme fromthe native source (29). Because these products can be obtained from anon-sterile, low input, renewable and easily scalable source, the costsof synthesis in plants are negligible. We confirmed the performance andcontainment of the vectors in four field trials (1991, 1994, 1995,1996).

The vectors of the invention are based on chimeras between the 6.4 kbsingle-stranded RNA genome of tobacco mosaic virus (TMV) and othermembers of the tobamovirus group. Most of the TMV genome encodesoverlapping reading frames required for replication and transcription(FIG. 1A). These are located at the 5′ end of the virus and translatedfrom genomic RNA yielding proteins of 126 and 183 kDa. Expression of theinternal genes was controlled by different promoters on the minus-senseRNA that direct synthesis of 3′-coterminal subgenomic mRNAs producedduring replication. The 30 kDa protein, which was required for the virusto move from cell to cell, was produced early and in relatively lowamounts, whereas the 17.5 kDa coat protein was produced late and usuallyas the most abundant protein in infected cells. Largely because of thestrength of the coat protein subgenomic promoter, during peak proteinsynthesis the coat protein can be produced at up to 70% of the totalrate of cellular protein synthesis without appreciably reducing hostprotein synthesis (30).

The entire cDNA of the TMV genome was cloned behind a bacterial phagepromoter in an E. coli plasmid. Precise replicas of the virion RNA canbe produced in vitro with RNA polymerase and dinucleotide cap, m⁷ GpppG.This not only allows manipulation of the viral genome for reversegenetics, but it also allows manipulation of the virus into a genetransfer vector. Subgenomic promoters from divergent viral strains canbe added to the genome to direct the expression of foreign genes.Enormous quantities of mRNA are synthesized and delivered directly fromthe cytoplasm to the ribosome. TMV-based transient vectors offersignificant advantages over integration of genes into plant chromosomes.By altering the molecular exclusion limits of the cellular junctionsbetween adjacent plant cells, the vector invades virtually every cell ofthe plant during a period of 2 weeks post-inoculation. For many geneproducts, the recombinant protein accumulates to several percent of thetotal protein during this brief period of time. In contrast, it was verytime consuming and labor intensive to generate, select, and breedtransgenic plants for recombinant protein production. Many of theseselections were culled because of poor expression due to positioneffects or gene silencing phenomena. In many more lines, the levels ofproduct accumulation was too low for development of a viable commercialprocess.

EXAMPLE 1

We have established that a recombinant human lysosomal enzyme (rGCB)synthesized in transgenic tobacco has comparable activity to the sameenzyme isolated from other native and recombinant sources. We alsoinvestigated the feasibility and economic advantages of purifying largequantities of active rGCB from plants. We designed and fabricatedlaboratory equipment that enabled us to optimize the key initial stepsof a purification process in the laboratory using kilogram quantities ofbiomass from our greenhouses. We standardized a series of assays forsecretory and intracellular marker enzymes in addition to rGCB assaysthat allowed us to monitor both lab and field expression as well as thepurification process. Leaf tissue was infiltrated with a suitableextraction buffer while submerged in a large vacuum chamber, allowingthe solution to reach the leaf intercellular fluid containing rGCB. TheIF fraction was recovered by centrifugation in a custom collectionchamber and “basket” centrifuge rotor compatible with a conventionalBeckman J2-21 spindle. rGCB was trapped from the dilute IF solution byexpanded bed adsorption chromatography using a hydrophobic resin andeluted with polyethylene glycol. A second ion exchange chromatographystep was implemented for an overall yield of 1.7 mg/kg at 41% purity tothis stage. These procedures were then scaled-up to 100 kg duringseveral pilot-process experiments in a field trial using analogousindustrial bioprocess equipment. These results are summarized in thetable below. Three lots of rGCB were further purified by RP-HPLC andused for carbohydrate profiling and composition analysis. In NMRexperiments we confirmed that the GCB from the plant IF contains anN-linked glycan previously reported to occur in glycoproteins isolatedfrom plant seeds and tissue cultures. This type of chain contains theplant-specific carbohydrate linkages of α 1-2 xylose and β 1-3 fucose onthe trimannosyl core.

Making Transgenic Tobacco Plants to Produce Glucocerebrosidase

Several founder plant lines for genetically stable expression of rGCBwere generated and characterized. Under greenhouse conditions individualplants accumulate rGCB to at least 1.3% of the total protein in the leafintercellular fluid as estimated from enzymatic assays. This representsa 50-fold enrichment relative to the crude lysosomal fraction ofplacental extracts used as the starting material for the productCeredase™.

Transgenic Tobacco Leaves Express Moderate Levels of rGCB. We combined adual promoter from Cauliflower Mosaic Virus (35S), a translationalenhancer from Tobacco Etch Virus and a polyadenylation region from thenopaline synthetase gene of Agrobacterium tumefaciens with the nativehuman GCB cDNA to create plasmid pBSG638 (33; FIG. 14). These expressionelements are widely used to provide the highest possible constitutiveexpression of nuclear encoded genes. Depending on the nature ofindividual proteins, these vectors can be used to accumulate moderatelevels of recombinant proteins in most tissues of many plant species.

Using a standard Agrobacterium-mediated transformation method, weregenerated 93 independent kanamycin-resistant transformants from leafdiscs of four different tobacco cultivars (the T0 generation). InWestern blots of total protein extracts, cross-reacting antigen wasdetected in 46 of these T0 individuals with antibody raised againsthuman glucocerebrosidase. Specificity of the plant-expressed recombinantenzyme was confirmed by hydrolysis of ¹⁴C-radiolabeled glucosylceramide.

Leaf Disc Transformation with Agrobacterium tumefaciens (59, 60, 61)Method

-   -   1) Transform the T-DNA plasmid into A.t. LBA4404 selecting for        the bacterial Ab^(R) gene (generally Km at 100 ug/ml).    -   2) Pick a single colony into YEB medium plus antibiotic and grow        at 28° C. overnight (to saturation; often takes a little longer        than overnight).    -   3) Take aseptic or surface-sterilized Nicotiana tabacum (MD609,        Xanthi, SR1, Samsun) leaves, remove midrib and cut into leaf        “chunks” ˜1 cm².    -   4) With sterile forceps, dip (submerge) the leaf disc into the        Agrobacterium suspension.        -   Placing the bacterial culture into a small petri dish is            convenient.    -   5) Remove the leaf disc from the Agrobacterium and place the        disc on regeneration medium. Place the discs so that the        underside of the leaf is up. (They seem to do better this way,        perhaps because of better gas transfer.)        -   Use needle-nose forceps to handle the discs, thus            introducing small puncture wounds into which Agrobacterium            can infect; small wounds are good, major damage (e.g.,            crushing) to the disc is bad.    -   6) Seal plate containing discs with Parafilm® and incubate at        25-28° C., preferably in light with a yellow filter to inhibit        UV degradation of the medium.    -   7) After 2 days co-incubation, transfer the leaf discs to        selective plates (regeneration medium plus 500 ug/ml        Cefotaxime).    -   8) After 2 more days, transfer discs to regeneration medium plus        500 ug/ml cefotaxime and 100 ug/ml kanamycin    -   9) When normal-looking shoots appear, excise them, taking care        not to excise any callus, and place in rooting medium.        -   Callus on the end of the stem generally prevents rooting,            and could lead to a chimeric set of shoots.        -   The lower % agar makes it easier to wash the agar off the            roots when transferring to soil.        -   If there is time, it is a good practice (when the plants are            rooted and growing) to cut the shoots off and re-root them.            Escapes will generally not root on Km medium.    -   10) When roots first appear, remove plantlets, wash agar from        the roots and plant in soil medium in small pots. Cover pots        with a plastic bag for the first 5 days or so to retain humidity        and reduce transplantation shock.

-   1 Liter of Regeneration Medium contains:

-   MS Salts

-   30 g sucrose

-   1 ml of 0.5 mg/ml nicotinic acid

-   1 ml of 0.5 mg/ml pyridoxine HCl

-   2 ml of 0.5 mg/ml thiamine HCl

-   2 ml of 50 mg/ml inositol

-   1.5 ml of 0.1 mg/ml IAA

-   5.0 ml of 1.0 mg/ml 2-IP-2-iminopurine

-   8 g of agar, pH 6.0

-   1 Liter of Rooting Medium contains:

-   ½, ×MS Salts

-   10 g sucrose

-   2 ml of 0.1 mg/ml IAA

-   8 g agar, pH 5.7

A deposit at ATCC under the Budapest treaty was made on Jul. 25, 2000 ofseed from Nicotiana benthamiana MD609, Accession No. PTA 2258.

According to these expression results the rGCB positive transformantswere ranked into moderate (A), low (B) and negligible (C) activitygroups (Table 1).

TABLE 1 EXPRESSION OF rGCB IN THE T0 GENERATION. Number of SpecificActivity Group Individuals Units/mg A 13 130-390 B 20  70-130 C 13 24-68Controls  8  0-10 Specific Activity is based on hydrolysis of [14C]-glucosylceramide (Units = nmol/hr).

Plant rGCB is Similar to Macrophage-Targeted Glucocerebrosidase. Wefound reaction conditions to preferentially inhibit rGCB enzyme activityin the presence of plant glucosidases using the suicide substrateconduritol B-epoxide (CBE). Total glucosidase activity, and rGCBactivity were measured by hydrolysis of the fluorescent substrate4-methylumbelliferylglucopyranoside (4-MUG) with and without CBE. Totalprotein was determined by the method of Bradford. Detergents arenecessary to solubilize and stabilize activity of thismembrane-associated enzyme. Using a small scale (˜100 mg fresh weight)extraction procedure, several detergents were compared for yield ofenzyme activity and purity (including; IGEPAL CA-630, Tween-20,Tween-80, Triton X-100, Triton X-114, CHAPS, taurocholic acid, cholicacid, deoxycholate and taurodeoxycholate). Buffer without detergent,deoxycholate, taurocholate and cholate below their critical micelleconcentrations (CMC) (0.1%) yielded low units of rGCB. All of the otherdetergents gave comparable specific activity and yields of totalactivity with Tween-80 yielding slightly higher activity. The dialyzablebile salt, sodium taurocholate and the lower CMC detergent Tween-80 werecompared at a range of concentrations (0.1-1% and 0.001-1%,respectively). Tween-80 at 0.15% and taurocholate at 0.5% gave the bestyield and purity.

A number of chromatography steps were evaluated for purification of rGCBfrom total homogenates (Table 1). As is the case for the nativeplacental enzyme, hydrophobic resins provide the most significantpurification gains. Gel filtration, Con A Sepharose and affinitychromatography also worked very well, but some of these approaches maybe impractical on a large scale. Both anion and cation chromatographymay prove useful, but the ideal buffer conditions for stabilization ofenzyme activity remain to be determined.

TABLE 2 SUMMARY OF CHROMOTOGRAPHY RESULTS Column Matrix Type ResultsOctyl Sepharose 4 FF Hydrophobic + Phenyl Sepharose HP Hydrophobic +Phenyl Sepharose 6 FF Hydrophobic + Butyl Sepharose 4 FF Hydrophobic −Alkyl Superose Hydrophobic − SP Sepharose FF Strong Cation +/−− QSepharose FF Strong Anion +/−− Con A Sepharose Lectin Affinity +/−NHS-Activated Sepharose HP Antibody Affinity + Sephacryl S-100 HR GelFiltration + (+) Effective increase in Specific Activity; (+/−) Needsenzyme activity stabilized; (+/−−) Variable results; (−) Poor binding.

The post-translational processing of native glucocerebrosidase (GCR) inhuman cells is complex. Two primary translation products are derivedfrom two in-phase start codons. These precursors, a 2:1 mixture of 60kDa and 57 kDa proteins, are proteolytically processed to 55 kDa as theypass into the lumen of the ER. High mannose and complex glycans aresubsequently added in the ER and Golgi compartments to yield 62 and 66kDa glycoforms. Finally, exoglycosidases generate a mature 59 kDalysosomal enzyme. Recall that glycosylation is required for bothenzymatic activity and lysosomal targeting of transfused enzyme. Sialicacid, galactose, and N-acetylglucosamine residues are enzymaticallyremoved in vitro by the sequential action of glycosidases to prepareglucocerebrosidase for therapy. The core pathway for biosynthesis andprocessing of N-linked complex glycans in plants appears identical tothat found in animals. There are three known differences which occurlater in the pathway. Sialic acid is not reported in complex glycansfrom plants, and the α1-3 fucose and β1-2 xylose linkages are unique(34). As analyzed by SDS/PAGE, rGCB has an apparent molecular weight of59 kDa, and comigrates with the mannose-terminal therapeutic glycoform.We have not yet detected a significant shift in mobility upon treatmentwith glycosidases (PNGase F, Endo H, α1-3 fucosidase) in our preliminaryglycosylation analysis. However, the enzyme has an apparent molecularweight increase of 4 kDa over the proteolytically processed andunglycosylated form (55 kDa) and must be glycosylated for activity.Additional digestions are in progress with a more extensive set of endo-and exoglycosidases and known plant glycoprotein controls. N-GlycosidaseA is reported to hydrolyze all types of N-glycan chains fromglycopeptides and glycoproteins.

The signal peptide of rGCB is processed at the correct site. A verysmall quantity of protein was prepared for sequence analysis bypurification through Phenyl-Sepharose, ConA-Sepharose and RP-HPLC toproduce a single band on SDS-PAGE comigrating with authenticglucocerebrosidase. The sequence obtained was consistent with the knownsequence of processed GCR (Table 3). In this particular analysis, thefirst two positions were not resolved because some degradation occurredduring sample preparation. Correct proteolytic cleavage of a signalpeptide is also confirmed for a mouse antibody light chain moleculeexpressed in tobacco leaves (35).

TABLE 3 STRUCTURE OF THE N-TERMINUS OF rGCB N-terminal Amino AcidSequence X X P X I P K S F G Y rGCB from tobacco (SEQ ID NO: 35) A R P CI P K S F G Y GCR human (SEQ ID NO: 36)Plant rGCB Accumulates in the Leaf Intercellular Fluid. We localizedrGCB to the intercellular fluid of the leaf using the following simpleexperimental design. Leaves were removed from the plant at the petioleand slit down the midrib into two equal halves. To obtain a totalcellular homogenate, one group of half-leaves was ground in the presenceof 4 volumes of detergent extraction buffer (100 mM potassium phosphatepH 6, 5 mM EDTA, 10 mM □-mercaptoethanol and 0.5% w/v sodiumtaurocholate) with a mortar and pestle. To recover the IF, the sameenzyme extraction buffer was infiltrated into the opposing group ofhalf-leaves by submerging the tissue and applying moderate vacuumpressure. After draining off excess buffer, the undisrupted half-leaveswere rolled gently in Parafilm, placed in disposable tubes and the IFcollected by low-speed centrifugation. The IF fraction is quite clearand non pigmented and can be applied directly to Phenyl Sepharosehydrophobic resin.

The results of a typical experiment are shown in Table 4. The increasein specific activity corresponds to a similar increase in the amount ofcross-reacting material observed in a Western blot and is therefore notan artifact of the enzyme assay in the different fractions. Furthermore,rGCB activity was very stable in crude extracts using this particulardetergent buffer. The increase in specific activity can therefore beattributed to an enrichment of rGCB in the IF relative to the whole cellhomogenate. The actual concentration of rGCB in the IF is likely to bemuch higher, because PAGE analysis of the IF fraction shows somecontamination with known cytoplasmic markers. The highest specificactivity we have measured in an IF sample is 20,000 U/mg. If we assumerGCB has the same specific activity as the human enzyme (1.5×10⁶ U/mg),this corresponds to 1.3% of the IF protein obtained by this method.

TABLE 4 LOCALIZATION OF rGCB TO THE INTERCELLULAR FLUID Fresh TotalProtein Total Protein rGCB Total rGCB Specific % Recovery Weight VolumeConc. Protein Yield Conc. rGCB Yield Activity rGCB X-Fold Sample (gr)(ml) (mg/ml) (mg) (mg/gr) (U/ml) (U) (U/gr) (U/mg) in IF PurificationIntercellular Fluid 2.48 1.9 0.24 0.45 0.18 720 1368 552 3007 22 18Homogenate 2.08 8.1 3.89 31.48 15.13 653 5289 2543 168Specific activity is based on the hydrolysis of 4-MUG inhibited by 0.5mM CBE. Units (U)=nmol/hr. Because the amount (in nanograms) of crossreacting material observed in a quantitative Western blot correspondswithin experimental error to the amount (in nanograms) of enzymecalculated on the basis of activity, we believe the plant rGCB wassynthesized with high specific activity. This was a very important andfavorable indirect estimate of specific activity. The enzyme waspurified to homogeneity to measure more precisely the actual specificactivity.

High Levels of rGCB Expression in Leaf Tissue Induce Gene Silencing. TheT0 individuals described in Table 4 are by definition hemizygous. Theycontain various loci generated from independent insertion events, havingno corresponding insert on the homologous chromosome. The thirteen T0individuals from Group A were self-pollinated and assayed for levels ofenzyme expression in the T1 generation in order to analyze the effectsof gene dosage (homozygotes versus hemizygotes) and to identifycandidate T1 families for future seed increase. Kanamycin-resistanttransgenic plants were randomly selected from segregating families andanalyzed for rGCB expression. The number of probable loci was estimatedby chi-square analysis of the linked kanamycin-resistant phenotypeat >95% confidence level. There are several T1 families with a heritablemean rGCB activity in the range of 200-300 U/mg (nmol 4-MUG hydrolyzedper hour) in the total homogenates that we have selected for furtherproduction of the enzyme (Table 5).

TABLE 5 EXPRESSION OF rGCB IN THE T1 GENERATION Mean Tobacco T1 NumberSpecific Activity Standard Number of Cultivar Family of Loci Units/mgError Individuals Samsun 963 2 294 25 23 Samsun 881 1 242 22 16 MD609920 1 205 15 38 Xanthi 902 1 202 17 5 Samsun 883 1 201 18 13 Xanthi 8321 195 18 9 SR1 826 1 184 16 40 SR1 834 1 145 9 32 Xanthi 851 1 140 15 5Samsun 837 1 129 16 8 Xanthi 831 1 114 21 10 Xanthi 833 1 107 12 5Xanthi 807 1 to 2 87 12 9 Controls 40 8 20However, of 235 T1 plants analyzed, the single individual having thehighest activity and the single observation of completely nullexpression were siblings of the T1 family 826. Moreover, extracts from826 were also quantitatively the second highest sample of the original46 analyzed for enzyme activity in the T0 generation. By Western blot,we analyzed protein extracts from several T1 siblings of this family,including the highest (612 U/mg) and the lowest (0 U/mg) and found aclear linear correlation between the amount of cross-reacting protein at59 kDa and the activity loaded in each lane. In addition to the 59 kDaband, there were also variable amounts of cross-reacting protein at 52kDa. In the null individual there was only the 52 kDa protein. We neverobserved this molecular weight species in the T0 extracts or in anyother T1 family. There was no evidence of proteolytic activity in thissample as judged by mixing the null sample with high activity extractsand analyzing by enzyme assays and Western blots after incubation at 37°C. If the apparently truncated rGCB was derived from proteolyticcleavage, the protease activity must be both physiologically induced andinactive under these isolation conditions. When the null individual wasself-pollinated and the T2 generation analyzed, enzyme expressionreappeared as in the T1 and T0. Our working hypothesis was that thetobacco plant is able to limit the expression of the foreign enzyme asconstitutively expressed from this cDNA construct, and that thethreshold for the stochastic induction of this response duringdevelopment occurs at an expression level corresponding to approximately600 U/mg specific activity in the crude homogenate. Of the lines wecreated, 826 were able to produce enough mRNA to exceed this thresholdin the homozygous state.

The silencing of genes in plants is a recently described phenomenon.Work has been done detailing a cellular surveillance mechanism that hasapparently evolved to specifically degrade excess RNA (36). In one case,specific RNA cleavages near the 3′-end of the transcript initiate theremoval of the transcript. Our description of the silencing of rGCBabove 600 U/mg is the first association of silencing with a truncatedprotein, and may well be caused by a specific mRNA (and not protein)cleavage event. Gene silencing may determine an upper limit ofexpression attainable using constitutive transgene expression.

We subcloned the cDNA for glucocerebrosidase into a TMV-transient vectorand cDNA combinations. Transcripts were synthesized in vitro andinoculated directly onto lower leaves of whole plants. In each case,there was an additional lag time of about 2 weeks post-inoculationbefore appearance of virus in the upper leaves of the plant and in eachcase the viral population no longer carried a significant portion of thegene. We detected no significant enzyme activity in either inoculated orsystemically infected leaves. Very recently, we detected the gene inroot tissue and in transfected protoplasts. There appears to be anincompatibility with leaf expression under conditions of viralamplification of the rGCB mRNA. This incompatibility selects for loss ofthe sequence from the viral population.

To further investigate the nature of the leaf incompatibility with rGCBexpression, we built the construct pBSG641. This plasmid contains therGCB gene substituted into the coat protein region. The remainingportion of the entire genome was then placed under control of the 35Spromoter. The promoter was designed to initiate RNA synthesis such thatthe correct 5′-end of TMV would be synthesized. A custom-designed,self-cleaving ribozyme sequence positioned at the end of the genomeyields a native 3′-end upon cleavage. The vector was designed forsynthesis of infectious transcripts in vivo from a chromosomallyintegrated locus and production of rGCB through viral amplification ofsubgenomic mRNA in the cytoplasm. The vector alone without the gene forrGCB produces a systemic but capsid- free, “naked-RNA” infection (38).This RNA co-suppression is the subject of issued U.S. Pat. No. 5,922,602issued Jul. 13, 1999, the disclosure of which is incorporated herein byreference.

We introduced the construct depicted in FIG. 15 into Agrobacterium andtransformed tobacco plants as described above. In this case many of theplant leaves displayed necrotic lesions as transfection events randomlyoccurred during growth and development and expansion of leaves. Theselesions never formed on control transformed plant lines containingvector only sequences capable of replication. These lesions wereidentical in appearance to the types of lesions induced by plantpathogens during a type of disease resistance reaction, termed thehypersensitive response (HR). Therefore, under conditions where weexpect to accumulate large quantities of active enzyme, an HR issignaled by some component of the vector infection specific to rGCB.There are very few of these so-called HR “elicitors” characterized inthe literature. Possibly the rGCB enzyme itself, or a secondarymetabolite resulting from enzymatic activity, or even rGCB RNA, mayinduce the HR. In any case, we hypothesize that the HR selects for lossof the gene from the viral RNA population. It is important to rememberthat this is not a simple genetic instability phenomenon. Underconditions where an HR is not induced, we have synthesized many proteinsusing TMV-based RNA viral vectors to levels of several percent of thetotal soluble cell protein without loss of the inserted gene even aftervirion passage.

Expression of rGCB in Transgenic Tobacco is Robust. In severalexperiments, we inoculated wild type TMV onto rGCB containing transgenictobacco and found a ˜1.5-2 fold increase in the specific activity oftotal homogenates. It appears that the viral infection causes anincrease in promoter activity, and/or the secretion and accumulation ofactive enzyme. This was an important result, because it demonstratesthat the expression was compatible with a TMV infection, aphysiologically severe stress condition. Furthermore, in separate work,we have used chimeric TMV particles as recombinant carriers for theproduction of small peptides (31).

Conclusions

We used a wide range of gene expression tools to investigate theaccumulation of rGCB in mature tobacco plants. Our results suggestattractive yield, quality and cost objectives can be met with furtherdevelopment. We observed two independent phenomena currently limitingthe accumulation of enzyme activity in whole plants; gene silencing inone transgenic line, and a plant leaf hypersensitive response totransient vector mediated synthesis.

These current limitations in gene expression only serve to underscorethe advantages and utility of agriculture for recombinant proteinproduction. We have generated several transgenic tobacco lines as areliable source of biomass for the production of high specific activityenzyme. Because the biomass is accumulated under no sterile growthconditions and production is inexpensively scaled to the quantitiesdesired, it becomes feasible to exploit a dilute but enriched sourcesuch as the intercellular fluid fraction for industrial processdevelopment. This contrast is most clearly summarized in Table 6.

TABLE 6 INITAL STEPS IN THE PURIFICATION OF GLUCOCEREBROSIDASE PLACENTAHOMOGENAT TOBACCO HOMOGENATE TOBACCO LEAF IF Specific Specific SpecificPurification Activity Activity Recovery Activity Activity RecoveryActivity Activity Recovery Procedure Units/kg Units/mg % Units/kgUnits/mg % Units/kg Units/mg % Detergent 1,510,000 375 100 1,870,000 230100 877,000 9,967 100 Extraction Concentration/ 707,000 9,330 471,540,000 14,400 82 Delipidation Hydrophobic 554,000 147,000 361,242,000 82,000 74 535,000 34,547 61 Chromatography

The placental homogenate procedure is adapted from Furbish et al., (10)starting with a 14,000×g sedimented material. In a typical preparation15-30 kg of fresh placentas were processed. The tobacco homogenate isbased on the average of 2 typical 1 kg extractions of the leaf biomass.The IF data is from an average of 5 small scale extraction experiments(2-200 grams fresh weight), and a single chromatography run of an IFconcentrate. For comparative purposes all yields are normalized to 1 kg.

TABLE 7 GCB IF PILOT PROCESS Greenhouse Scale (1 kg) Field Scale (100kg) Specific Total Specific Total Purification Activity ActivityRecovery Purification Activity Activity Recovery Purification StepUnits/kg Units/mg % Fold Units/kg Units/mg % Fold IF 4,153,533 20,388100 1.0 434,927 2,745 100 1.0 Phenyl SL 3,738,180 147,813 91.4 7.25194,722 12,960 44.4 5.0 SP Big Beads 2,740,086 650,377 67.0 31.9 145,06099,220 33.1 38.2For comparative purposes all yields are normalized to 1 kg.

The greenhouse/laboratory scale process is based on an average of 2infiltration/chromatography runs starting with 1 kilogram of freshweight leaf tissue.

The GenBank accession No. for glucocerebrosidase is M11080. The fieldscale process is an average of 7 large scale infiltrations consisting of100 kilograms of fresh weight tissue. Enzyme activity is based on thecleavage of 4-methylumbelliferylglucoside (1 Unit=1 nmol/hr). One factorcontributing to a lower apparent yield in the field on a fresh weightbasis is that rGCB is concentrated in the leaf lamina and in the labscale procedure the midrib was removed.

Preparation of Solutions for GCB Assay with 4-Methylumbelliferylβ-D-glucopyranoside

1. GCB Assay Buffer

-   0.1 M Potassium Phosphate, 0.15% Triton X-100, 0.125% sodium    taurocholate (Sigma T-4009), 0.1% bovine serum albumin, 0.02% sodium    azide, pH 5.9

Dissolve 13.6 grams of potassium phosphate monobasic (KH₂PO₄) in 950 mlof distilled water. Add 1.25 g of sodium taurocholate and 1.5 g ofTriton X-100. Triton X-100 is a very viscous liquid and should beweighed rather than pipetted in order to achieve a reproducible buffer.Add 2 ml of 10% sodium azide and 1 gram of bovine serum albumin (BSA).Stir until all material has dissolved. Adjust the pH to 5.9 by theaddition of a small amount of 1 N NaOH, then bring up to 1000 ml withwater. Filter sterilize and store at 4° C. This buffer is stable formany months.

2. Stopping Buffer

-   0.1 M Glycine in 0.1 M NaOH

Dissolve 4 grams of NaOH and 7.51 grams of glycine in 1 liter ofdistilled water. Filter sterilize and store at 4° C. (Stable for yearsat 4° C.).

3. Substrate (Sigma M-3633) FW 338.3

-   15 mM 4-methylumbelliferyl β-D-glucopyranoside (4-MUG) in assay    buffer

Weigh out 1 gram of 4-MUG into a 500 ml Erlenmeyer flask. Add exactly197 ml of Assay Buffer (Substrate dilution Buffer) and heat in a hotwater bath to dissolve. Caution: Heating too aggressively results inunacceptably high background fluorescence. After cooling, dispense into5-7 ml aliquots in 15 ml polypropylene tubes, let tubes cool to roomtemperature and freeze at −20° C. for later use.

4. 125 mM Conduritol β-epoxide (CBE) (Toronto Research C-66600)

MW=162.18

Dissolve 100 mg of CBE in 4.92 ml of 0.1 M KPO₄ Buffer, pH 6.0. Dispenseinto 200-500 μl aliquots and store at −20° C.

5. 0.1 M KPO₄ Buffer, pH 6.0 1.75 ml of 0.5 M KH₂PO₄ (Solution A) 87.5mM 0.246 ml of 0.5 M K₂HPO4 (Solution B) 12.3 mM Add distilled water to10 ml.

Reagents: Potassium Phosphate Monobasic (KH₂PO₄) Fisher Scientific P285Potassium Phosphate Dibasic (K₂HPO₄) Fisher Scientific P288 Triton X-100Sigma X-100 Sodium Taurocholic Acid Sigma T-4009 Bovine Serum Albumin,Fraction V Sigma A-2153 Sodium Azide Sigma S-2002 Glycine Sigma G-4392Sodium Hydroxide Pellets Fisher Scientific S318 Conduritol β-epoxide(CBE) Toronto Research C-66600 MW = 162.18 4-Methylumbelliferylβ-D-glucopyranoside Sigma M-3633 (β-D-glucoside)

GCB Assay with 4-Methylumbelliferyl β-D-glucopyranoside (MUG)

1.0 Purpose

To measure the amount of glucocerebrosidase activity from transgenictobacco plants following infiltration and/or homogenization of thetissue. Measurement of fluorometric activity requires an accuratedetermination and relationship between fluorescence of the releasedmethylumbelliferone and its concentration under the assay conditions.

Scope

This is an inhibition assay. CBE inhibits human GCB. The fluorescentvalue used to calculate activity is based on the difference in valueswith and without inhibitor present. The fluorescent value with CBE(plant glucosidase) is subtracted from the fluorescent value withoutinhibitor which is both plant and human GCB activity. The differencebeing the value for human GCB expressed in the transgenic plants. Theassay is carried out using 5 μl of sample with 45 μl assay buffer +/−CBE at 37° C. This means that 2 tubes are needed per sample. Thisprocedure is applicable to the Glucocerebrosidase assay procedurerequiring a methylumbelliferone standard curve.

Equipment

-   Fluorometer (St. John Associates Fluoro-Tec 2001A with KV-418 filter    and 365 nm interference filter)-   10×75 mm cuvettes (St. John Associates)-   Water Bath-   Test Tubes 13×100 mm glass (VWR or equivalent)-   4-Methylumbelliferone (Sigma M-1381)-   Pipettes and pipette tips, 5 μl-1 ml (Rainin or equivalent)-   1.5 ml microfuge tubes    Precautions

The fluorometer should be warmed up for at least 20 minutes prior toreading samples. The power switch should be left on at all times. If thepower switch was turned off it may take longer (up to 1 hour) for theinstrument to stabilize.

Be certain to put away all reagents under proper storage conditionsafter reading assays. Any left over fluorescent substrates and CBE stockshould be returned to −20° C. The fluorescent substrate and CBE stockcan be frozen and thawed numerous times without any breakdown of thereagents. Do not save any substrate or assay buffer to which you addedCBE. These should be discarded. You should only make up enough reagentwith the inhibitor (CBE) that you currently need.

Procedure

-   -   1. Turn on the water bath and check to be certain the        temperature is set to 37° C.    -   2. Turn on fluorometer to warm up by flipping up the PMT switch.        It should be turned on at least 15-20 minutes before taking        readings. If the power switch was turned off it may take longer        (up to 1 hour) for the instrument to stabilize.    -   3. Remove assay buffer from refrigerator and CBE from −20° C.        freezer. Thaw CBE on ice.    -   4. Defrost the appropriate amount of methylumbelliferyl        β-D-glucopyranoside (MUG) substrate. You need 400 μl of MUG for        each sample (+/−CBE). Place tubes in 37° C. H₂O bath for        approximately 10 minutes to get MUG into solution. Note: There        may be a small amount of insoluble material (MUG) in each tube        even after the 10 minutes at 37° C. Vortex before use. Keep at        room temperature until ready to use.    -   5. Remove enough GCB Assay Buffer from the stock bottle that        will be needed for your samples and transfer to a 15 ml tube.        Add appropriate amount of 125 mM stock solution of CBE to assay        buffer so the final concentration is 0.55 mM CBE. CBE stock is        stored at −20° C. Thaw some CBE on ice now if you have not        already done so. The assay buffer +CBE may be kept at room        temperature if assays will be completed within 1 hour otherwise        store solutions with CBE on ice.

Note: you want the final conc. of CBE in the assay to be 0.5 mM, you areadding 45 μl of buffer to 5 μl of sample so the starting conc. of CBEshould be 0.55 mM. Example: Add 8.8 μl of 125 mM CBE stock solution to1.991 ml of assay buffer to equal 0.55 mM CBE in assay buffer. This isenough buffer to carry out at least 40 assays.

-   -   6. Label two 13×100 mM glass tubes for each sample to be assayed        with a number and the same number and a “+” sign. (1, 1+, 2, 2+,        etc). Tubes with “+” contain CBE, tubes with just a number will        not have CBE added to the assay buffer or MUG.        Carry out the following steps on ice.    -   7. Place numbered tubes in white racks and place in Styrofoam        ice chest filled with enough ice and H₂O (Ice Bath) to cover the        volume of fluid in these tubes.    -   8. Add 45 μl assay buffer to all the tubes with numbers only.        -   Add 45 μl of assay buffer+CBE to all the “+” tubes.        -   Place pipette tip in bottom of tube to deliver assay buffer            to bottom of tube.    -   9. Remove metal tip ejector from P-10 pipetman (so pipetman will        reach bottom of tube) and pipette 5 μl of sample into each set        of appropriate tubes of assay buffer (one “+” and one tube with        a number only (−CBE) for each sample or twice this number if        done in duplicate). Place pipette tip in bottom of tube to        deliver sample directly into assay buffer. This is very        important since you will be pipetting small volumes into these        tubes.        Be sure to include a Buffer sample to blank the fluorometer.        This will contain 5 μl of the same buffer that the samples are        in.

Note: See Dilution of Enzymes if your sample is too concentrated to readin the fluorometer. Basically, dilute your sample 1:5, 1:10, etc. inassay buffer, mix well, pulse in microfuge and add 5 μl of dilutedsample to assay buffer above. You should only need 5-10 μl of yoursample for the dilutions.

-   -   10. Incubate tubes at 37° C. for 10 minutes then place tubes        immediately on ice.    -   11. Aliquot the volume of MUG needed for “+” tube assays (200 μl        per assay+CBE) into a 15 ml polypropylene tube and add CBE stock        to a final concentration of 0.5 mM.        -   −4 μl of 125 mM CBE per 1 ml MUG in assay buffer=0.5 mM CBE    -   12. Add MUG+/−CBE to appropriate tubes.        -   Add 200 μl of MUG without CBE to all the tubes with a number            only (−CBE).        -   Add 200 μl of MUG+CBE to all the “+” tubes.        -   Place a plastic cap over each tube. Vortex sample and place            back on ice.    -   13. Transfer all tubes to 37° C. H₂O bath. Incubate at 37° C.        for 15 minutes with shaking. Set the shaker between the #4-6        settings.    -   14. Transfer tubes to ice bath. Quickly add 1 ml of stopping        solution to each sample. Remove rack of samples from ice.    -   15. You are now ready to read samples in fluorometer.    -   16. Be certain to put away all reagents under proper storage        conditions after reading assays. Any left over fluorescent        substrates should be returned to −20° C. The fluorescent        substrate and CBE stock can be frozen and thawed numerous times        without any breakdown of the reagents. Do not save any substrate        or assay buffer to which you added CBE. These should be        discarded. You should only make up enough reagent with the        inhibitor (CBE) that you currently need.        Dilution of Samples for Assays

Routine dilutions of samples should be carried out on ice using the GCBAssay Buffer described above to maintain enzyme activity. Generally a1:5 or 1:10 dilution of the sample is sufficient. Dilutions should becarried out in a microfuge tube. (Example: 1:10 dilution: 5 μl of samplein 45 μl of assay buffer, mix well and pulse sample in microfuge tobring all of the sample to the bottom of the tube). You should only need5-10 μl of your sample for the dilutions.

EXAMPLE 2

Extraction of Glucocerebrosidase Protein

Glucocerebrosidase (GCB), either derived from human placental tissue ora recombinant form from Chinese hamster ovary cells (CHO), is presentlyused in an effective but costly treatment of the heritable metabolicstorage disorder known as Gaucher disease. We combined a dual promoterfrom Cauliflower Mosaic Virus (35S), a translational enhancer fromTobacco Etch Virus and a polyadenylation region from the nopalinesynthetase gene of Agrobacterium tumefaciens with the native human GCBcDNA to create plasmid pBSG638. These expression elements are widelyused to provide the highest possible constitutive expression of nuclearencoded genes in plants. The CaMV promoter is further inducible bystress or wound treatment.

Using a standard Agrobacterium-mediated transformation method, weregenerated 93 independent kanamycin-resistant transformants from leafdiscs of four different tobacco cultivars (the TO generation). InWestern blots of total protein extracts, cross-reacting antigen wasdetected in 46 of these TO individuals with antibody raised againsthuman glucocerebrosidase. Specificity of the plant-expressed recombinantenzyme was confirmed by hydrolysis of 14C-radiolabeled glucosylceramide.According to these expression results the rGCB positive transformantswere ranked into moderate (A), low (B) and negligible (C) activitygroups.

We also found reaction conditions to preferentially inhibit rGCB enzymeactivity in the presence of plant glucosidases using the suicidesubstrate conduritol B-epoxide (CBE). Total glucosidase activity, andrGCB activity were measured by hydrolysis of the fluorescent substrate4-methylumbelliferylglucopyranoside (4-MUG) with and without CBE. Leavesfrom plants transfected with the vector TT01A 103L were removed at thepetiole and slit down the midrib into two equal halves. To obtain atotal cellular homogenate, one group of half-leaves was ground in thepresence of 4 volumes of detergent extraction buffer (100 mM potassiumphosphate p(I 6, 5 mM EDTA, 10 mM, B-mercaptoethanol and 0.5% w/v sodiumtaurocholate) with a mortar and pestle after freezing the tissue inliquid nitrogen. To recover the intercellular fluid (IF), the sameenzyme extraction buffer was infiltrated into the opposing group ofhalf-leaves by submerging the tissue and applying moderate vacuumpressure (500 mm Hg). After draining off excess buffer, the undisruptedhalf-leaves were rolled gently in Parafilm, placed in disposable tubesand the intercellular fluid (IF) was collected by low-speedcentrifugation (1,000 g). The weight of buffer recovered from theinfiltrated leaf tissue is recorded and varies from approximatelyone-half to equal the original weight of the leaf. GCB expression in IFextracts was quantified using a commercially available enzyme assayreagents and protocol. Total protein was determined by the methoddescribed by Bradford (Bradford, M. Anal. Biochem 72:248, 1976).

We have demonstrated that active recombinant GCB may be successfullyextracted from the intercellular fluid of plant leaves using the presentmethod. The GCB assay is based on MUG hydrolysis in the presence of CBE.The IF method results in a recovery of 22% of the total GCB activity ofthe leaf at a 18-fold enrichment relative to an extract obtained byhomogenization (H). The GCB production results may be improved byoptimizing the time post-inoculation with the viral vector andminimizing the contaminating viral coat protein from the intercellularfraction.

EXAMPLE 3

Laboratory Pilot Scale Purification of Glucocerebrosidase from theIntercellular Fluid of Tobacco

MD609 leaf tissue (1-2 kilograms) of transgenic tobacco expressing thelysosomal enzyme glucocerebrosidase was harvested, the mid vein removedand the tissue weighed. Tissue was submerged with 2-4 volumes of buffer(0.1 M KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5% taurocholic acid, 10 mMβ-mercaptoethanol) in an infiltration vessel that accommodates severalkilograms of leaf tissue at one time. A perforated metal plate wasplaced on top of tissue to weigh down the tissue. A vacuum of 25-27 in.Hg was applied for 1-2 minutes×3. The vacuum was released betweensubsequent applications. Tissue was rotated and the vacuum reapplied toachieve complete infiltration. Multiple applications of the vacuumwithout isolating the intercellular fluid constitutes a singleinfiltration procedure. An indication of complete infiltration is adistinct darkening in color of the underside of the leaf tissue. Excessbuffer on the tissue was drained. The intercellular fluid was releasedfrom the tissue by centrifuging the tissue in a basket rotor at 4200 RPM(2500×g) for 10 minutes. The intercellular fluid was collected using anaspirator hooked up to a vacuum pump (IF-1). Alternatively, the leaftissue can be re-infiltrated by placing the leaves back in theinfiltration vessel in the same buffer used above and the processrepeated (IF-2). The second infiltration does not require as manyapplications of the vacuum. Additionally, the buffer may be drained fromthe infiltration vessel (spent buffer) and pooled with the 1st and 2ndIF fractions. Collectively, IF-1, IF-2 and Spent Buffer constitutes theIF pool. The volume of intercellular fluid collected from theinfiltrated leaf tissue was between 50-100% of the leaf tissue by weightdepending on the number of infiltrations carried out.

Recombinant GCB was purified by loading the dilute intercellular (feedstream) directly on a Pharmacia Streamline 25 column containing PhenylStreamline resin. Expanded bed chromatography enabled us to capture,clarify and concentrate our protein in one step without the need forcentrifugation and/or microfiltration steps. The column was equilibratedand washed until UV-signal on recorder returned to baseline with 25 mMcitrate, 20% ethylene glycol, pH 5.0 and then eluted with 25 mM citrate,70% ethylene glycol. The eluted material was further purified on acation exchange resin, SP Big Beads (Pharmacia), equilibrated in 25 mMcitrate, 75 mM NaCl, pH 5.0. GCB was eluted with either a step gradientof 25 mM citrate, 0.5 M NaCl, 10% ethylene glycol, pH 5.0 or a lineargradient of 75 mM-0.4 M NaCl in 25 mM citrate, pH 5.0. Allchromatography steps were carried out at room temperature.

Using the suicide substrate, conduritol β-epoxide (CBE), inhibition ofrecombinant glucocerebrosidase (rGCB) activity in the presence of plantglucosidases was achieved. Enzyme activity was measured at 37° C. in areaction mixture containing 5 mM methylumbelliferyl β-D glucoside, 0.1 MPotassium Phosphate, 0.15% Triton-X100, 0.125% sodium taurocholate, 0.1%bovine serum albumin, pH 5.9 with and without CBE. Total glucosidaseactivity and rGCB activity were measured by hydrolysis of thefluorescent substrate 4-methylumbelliferyl glucopyranoside. One unit ofactivity is defined as the amount of enzyme required to catalyze thehydrolysis of 1 nmol of substrate per hour. Total protein was determinedusing the Bio-Rad Protein Assay based on the method of Bradford(Bradford, M. Anal. Biochem. 72:248; 1976).

Typically from 1 kilogram of leaves where IF-1 alone was collected weobtained 4 million unites of GCB at a specific activity of 20,000. TheUnits/kg increased to 6 million with a lower specific activity of 10,000when IF Pool was collected (IF-1, IF-2 and spent buffer). For moreinformation on these experiments, see U.S. Ser. Nos. 09/132,989 and09/500,554. The disclosures of which are incorporated herein byreference.

EXAMPLE 4

Ultrafiltration/Concentration of Intercellular Fluid from TobaccoExpressing Glucocerebrosidase

2.3 kilograms of MD609 leaf tissue from transgenic tobacco expressingthe lysosomal enzyme glucocerebrosidase was harvested, the mid veinremoved and the tissue weighed. Tissue was submerged with 2-4 volumes ofbuffer (0.1 M KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5% taurocholic acid, 10mM β-mercaptoethanol) in an infiltration vessel that accommodatesseveral kilograms of leaf tissue at one time. A perforated metal platewas placed on top of tissue to weigh down the tissue. A vacuum of 25-27in. Hg was applied for 1-2 minutes×3. The vacuum was released betweensubsequent applications. Tissue was rotated and the vacuum reapplied toachieve complete infiltration. Excess buffer on the tissue was drained.The intercellular fluid was released from the tissue by centrifuging thetissue in a basket rotor at 4200 RPM (2500×g) for 10 minutes. Theintercellular fluid was collected using an aspirator hooked up to avacuum pump (IF-1). The leaf tissue was re-infiltrated by placing theleaves back in the infiltration vessel in the same buffer used above andthe process repeated (IF-2). The buffer was drained from theinfiltration vessel (spent buffer) and pooled with the 1st and 2nd IFfractions. Collectively, IF-1, IF-2 and Spent Buffer constitutes the IFpool. The IF pool was filtered through Miracloth and then concentrated 6fold by passing the IF pool through a 1 sq. ft. spiral membrane (30Kmolecular weight cutoff) using an Amicon RA 2000 concentrator equippedwith an LP-1 pump.

Using the suicide substrate, conduritol β-epoxide (CBE), inhibition ofrecombinant glucocerebrosidase (rGCB) activity in the presence of plantglucosidases was achieved. Enzyme activity was measured at 37° C. in areaction mixture containing 5 mM methylumbelliferyl β-D glucoside, 0.1 MPotassium Phosphate, 0.15% Triton-X100, 0.125% sodium taurocholate, 0.1%bovine serum albumin, pH 5.9 with and without CBE. Total glucosidaseactivity and rGCB activity were measured by hydrolysis of thefluorescent substrate 4-methylumbelliferyl glucopyranoside. One unit ofactivity is defined as the amount of enzyme required to catalyze thehydrolysis of 1 nmol of substrate per hour. Total protein was determinedusing the Bio-Rad Protein Assay based on the method of Bradford(Bradford, M. Anal. Biochem. 72:248; 1976).

EXAMPLE 5

Pilot Scale Purification of Glucocerebrosidase from the IntercellularFluid of Field Grown Tobacco

100 kilograms of MD609 leaf tissue from transgenic tobacco expressingthe lysosomal enzyme glucocerebrosidase was harvested from the fieldeach day for a period of two weeks. The tissue was stripped off thestalks by hand and weighed. Five kilograms of leaves were placed intopolyester bags (Filtra-Spec, 12-2-1053) and four×5 kg bags of leaveswere placed into a metal basket. The metal basket containing the leafmaterial was placed in a 200 L Mueller vacuum tank containing ˜100liters of buffered solution (0.1 KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5%taurocholic acid, 10 mM β-mercaptoethanol). A 70 lb. stainless steelplate was placed over the leaves/bags to assure complete immersion. Avacuum was pulled 27 in. Hg, held for 1 minute and then rapidlyreleased. This vacuum infiltration was repeated for a total of twocycles. Multiple applications of the vacuum without isolating theintercellular fluid constitutes a single infiltration procedure. Anindication of complete infiltration is a distinct darkening in color ofthe underside of the leaf tissue. Following the vacuum infiltrations,the leaves and basket were removed from the vacuum tank. The bagscontaining the vacuum infiltrated leaves were allowed to gravity drainsurface buffer for ˜10 minutes, prior to centrifugation. Theintercellular fluid (IF) was recovered from the vacuum infiltratedleaves by centrifugation (1,800×g, 30 minutes) using a Heine basketcentrifuge (bowl dimensions, 28.0 inches diameter×16.5 inches).Collected IF was filtered through a 50 uM cartridge filter and thenstored at 4° C., until the entire 100 kilograms of tissue wasinfiltrated. This process was repeated with the next set of four 5 kgbags (5×20 Kg cycles total) until all the tissue was infiltrated.Additional buffer was added during each infiltration cycle to completelyimmerse the tissue. Alternatively, the leaf tissue can be re-infiltratedby placing the leaves back in the infiltration vessel in the same bufferused above and the process repeated (IF-2). Additionally, the buffer maybe drained from the infiltration vessel (spent buffer) and may be pooledwith the 1st and 2nd IF fractions. Collectively, IF-1, IF-2 and SpentBuffer constitutes the IF pool. The volume of intercellular fluidcollected from the infiltrated leaf tissue was between 42-170% of theleaf tissue by weight depending on the number of infiltrations carriedout.

Recombinant GCB was purified by loading the dilute intercellular (feedstream) directly on a Pharmacia Streamline 200 column containing PhenylStreamline resin. Expanded bed chromatography enabled us to capture,clarify and concentrate our protein in one step without the need forcentrifugation and/or microfiltration steps. The column was equilibratedand washed until UV-signal on recorder returned to baseline with 25 mMcitrate, 20% ethylene glycol, pH 5.0 and then eluted with 25 mM citrate,70% ethylene glycol. The eluted material was sterile filtered by passingthe eluted material through a 1 sq. ft. 0.8 um Sartoclean GF capsulefollowed by a 1 sq. ft. 0.2 um Sartobran P sterile filter (Sartorius,Corp.) and stored at 4° C. until the next chromatography step. Theeluted material from 4-5 days of Phenyl Streamline chromatography runswas pooled together and further purified on a cation exchange resin, SPBig Beads (Pharmacia), equilibrated in 25 mM citrate, 75 mM NaCl, pH5.0. GCB was eluted with a step gradient of 25 mM citrate, 0.4 M NaCl,10% ethylene glycol, pH 5.0. All chromatography steps were carried outat room temperature. The eluted material was sterile filtered by passingthe eluted material through a 1 sq. ft. 0.8 um Sartoclean GF capsulefollowed by a 1 sq. ft. 0.2 um Sartobran P sterile filter (Sartorius,Corp.) and stored at 4° C.

Using the suicide substrate, conduritol β-epoxide (CBE), inhibition ofrecombinant glucocerebrosidase (rGCB) activity in the presence of plantglucosidases was achieved. Enzyme activity was measured at 37° C. in areaction mixture containing 5 mM methylumbelliferyl β-D glucoside, 0.1 MPotassium Phosphate, 0.15% Triton-X100, 0.125% sodium taurocholate, 0.1%bovine serum albumin, pH 5.9 with and without CBE. Total glucosidaseactivity and rGCB activity were measured by hydrolysis of thefluorescent substrate 4-methylumbelliferyl glucopyranoside. Totalprotein was determined using the Bio-Rad Protein Assay based on themethod of Bradford (Bradford, M. Anal. Biochem. 72:248; 1976).

Typically from 1 kilogram of field grown tobacco, expressing GCB, whereIF-1 alone was collected we obtained 435,000 units of GCB at a specificactivity of 2,745. The Units/kg increased to 755,000 with a specificactivity of 3,400 when IF Pool was collected (IF-1, IF-2 and spentbuffer).

EXAMPLE 6

Total GCB (IF vs. Homogenate) in “GCB Field Test Virus”

100 kilograms of MD609 leaf tissue from transgenic tobacco expressingthe lysosomal enzyme glucocerebrosidase was harvested from the fieldeach day for a period of two weeks. The tissue was stripped off thestalks by hand and weighed. Five kilograms of leaves were placed intopolyester bags (Filtra-Spec, 12-2-1053) and four×5 kg bags of leaveswere placed into a metal basket. The metal basket containing the leafmaterial was placed in a 200 L Mueller vacuum tank containing ˜100liters of buffered solution (0.1 KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5%taurocholic acid, 10 mM β-mercaptoethanol). A 70 lb. stainless steelplate was placed over the leaves/bags to assure complete immersion. Avacuum was pulled 27 in. Hg, held for 1 minute and then rapidlyreleased. This vacuum infiltration was repeated for a total of twocycles. Following the vacuum infiltrations, the leaves and basket wereremoved from the vacuum tank. The bags containing the vacuum infiltratedleaves were allowed to gravity drain surface buffer for ˜10 minutes,prior to centrifugation. The intercellular fluid (IF) was recovered fromthe vacuum infiltrated leaves by centrifugation (1,800×g, 30 minutes)using a Heine basket centrifuge (bowl dimensions, 28.0 inchesdiameter×16.5 inches). Collected IF was filtered through a 50 uMcartridge filter and then stored at 4° C., until the entire 100kilograms of tissue was infiltrated. This process was repeated with thenext set of four 5 kg bags (5×20 Kg cycles total) until all the tissuewas infiltrated. Additional buffer was added during each infiltrationcycle to completely immerse the tissue. In order to evaluate how muchenzyme was recovered in the intercellular fluid, the tissue from whichthe intercellular fluid was isolated was then homogenized in a Waringblender with 4 volumes of the same infiltration buffer as above,centrifuged and the supernatant assayed for enzyme activity.

EXAMPLE 7

Chops Experiment

An experiment was carried out where 100 kilograms of MD609 leaf tissueof transgenic tobacco expressing the lysosomal enzyme glucocerebrosidasewas harvested off the stalks by hand, weighed and chopped into smallpieces to increase the surface area for buffer infiltration. Fivekilograms of leaves were placed into polyester bags (Filtra-Spec,12-2-1053) and four×5 kg bags of leaves were placed into a metal basket.The metal basket containing the leaf material was placed in a 200 LMueller vacuum tank containing ˜100 liters of buffered solution (0.1KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5% taurocholic acid, 10 mMβ-mercaptoethanol). A 70 lb. stainless steel plate was placed over theleaves/bags to assure complete immersion. A vacuum was pulled 27 in. Hg,held for 1 minute and then rapidly released. This vacuum infiltrationwas repeated for a total of two cycles. Following the vacuuminfiltrations, the leaves and basket were removed from the vacuum tank.The bags containing the vacuum infiltrated leaves were allowed togravity drain surface buffer for ˜10 minutes, prior to centrifugation.The intercellular fluid (IF) was recovered from the vacuum infiltratedleaves by centrifugation (1,800×g, 30 minutes) using a Heine basketcentrifuge (bowl dimensions, 28.0 inches diameter×16.5 inches).Collected IF was filtered through a 50 uM cartridge filter and thenstored at 4° C., until the entire 100 kilograms of tissue wasinfiltrated. This process was repeated with the next set of four 5 kgbags (5×20 Kg cycles total) until all the tissue was infiltrated.Additional buffer was added during each infiltration cycle to completelyimmerse the tissue. In order to evaluate how much enzyme was recoveredin the intercellular fluid, the tissue from which the intercellularfluid was isolated was then homogenized in a Waring blender with 4volumes of the same infiltration buffer as above, centrifuged and thesupernatant assayed for enzyme activity.

Recombinant GCB was purified by loading the dilute intercellular (feedstream) directly on a Pharmacia Streamline 200 column containing PhenylStreamline resin. The column was equilibrated and washed until UV-signalon recorder returned to baseline with 25 mM citrate, 20% ethyleneglycol, pH 5.0 and then eluted with 25 mM citrate, 70% ethylene glycol.All chromatography steps were carried out at room temperature Table 10below contains data from the chops experiment.

EXAMPLE 8

Pilot Scale of Purification of Alpha Galactosidase from theIntercellular Fluid of Nicotiana benthamiana.

Young actively growing Nicotiana benthamiana plants were inoculated withinfectious transcripts of a recombinant plant viral construct containingthe lysosomal enzyme α galactosidase gene. Systemically infected leaftissue (1-2 kilograms) was harvested from Nicotiana benthamianaexpressing α galactosidase 14 days post inoculation. The tissue wasweighed and submerged with 2-4 volumes of buffer (25 mM Bis Tris PropaneBuffer, pH 6.0, 5 mM EDTA, 0.1 M NaCl, 10 mM β-mercaptoethanol) in aninfiltration vessel that can accommodate several kilograms of leaftissue at one time. A perforated metal plate was placed on top of tissueto weigh down the tissue. A vacuum of 25-27 in. Hg was applied for 30seconds and then quickly released. The tissue was rotated and the vacuumreapplied to achieve complete infiltration which was confirmed by adistinct darkening in color of the underside of the leaf tissue. Excessbuffer on the tissue was drained. The intercellular fluid was releasedfrom the tissue by centrifuging the tissue in a basket rotor at 3800 RPM(2100×g) for 10-15 minutes. The intercellular fluid was collected usingan aspirator hooked up to a vacuum pump. In some instances only infectedleaf tissue was harvested. Alternatively, petioles and stems have beenharvested along with the leaf tissue for infiltration. The mid vein wasnot removed from the tissue prior to infiltration.

Alpha galactosidase was purified by loading the dilute intercellular(fed stream) directly onto a Pharmacia Streamline 25 column containingButyl Streamline resin. Expanded bed chromatography enabled us tocapture, clarify and concentrate our protein in one step without theneed for centrifugation and/or microfiltration steps. The column wasequilibrated and washed until UV-signal on recorder returned to baselinewith 25 mM Bis Tris Propane, pH 6.0 20% (NH₄)₂SO4 and then eluted with25 mM Bis Tris Propane, pH 6.0. The eluted material was further purifiedon Hydroxyapatite equilibrated with 1 mM NaPO₄ Buffer, 5% glycerol, pH6.0 and eluted with either a 1-250 mM NaPO₄ buffer, 5% glycerol, pH 6.0linear gradient or a step gradient. All chromatography steps werecarried out at room temperature.

Alpha galactosidase activity was measured by hydrolysis of thefluorescent substrate 4-methylumbelliferyl a-D galactopyranoside. Enzymeactivity was measured at 37° C. in a reaction mixture containing 5 mMmethylumbelliferyl a-D galactopyranoside, 0.1 M Potassium Phosphate,0.15% Triton-X100, 0.125% sodium taurocholate, 0.1% bovine serumalbumin, pH 5.9. Total protein was determined using the Bio-Rad ProteinAssay based on the method of Bradford (Bradford, M. Anal. Biochem. 72:248; 1976).

From 1 kilogram of leaves, we typically obtain between 140-160 millionunits of α galactosidase at a specific activity of 800,000 following asingle infiltration procedure (IF-1). Table 11 below contains data thatis representative of several experiments.

EXAMPLE 9

Pilot Scale Purification of Glucocerebrosidase from the IntercellularFluid of Field Grown Tobacco

Transgenic tobacco (MD609) expressing the lysosomal enzymeglucocerebrosidase was mechanically inoculated with a tobacco mosaicvirus derivative containing a coat protein loop fusion, TMV291, (Turpen,et.al., 1995, Bio/Technology 13: 23-57). A total of 100 Kg oftransgenic, transfected leaf tissue was harvested from the field, fiveweeks post inoculation. The tissue was stripped off the stalks by handand weighed. Five kilograms of leaves were placed into polyester bags(Filtra-Spec, 12-2-1053) and four×5 kg bags of leaves were placed into ametal basket. The metal basket containing the leaf material was placedin a 200 L Mueller vacuum tank containing ˜100 liters of bufferedsolution (0.1 KPO₄ buffer, pH 6.0, 5 mM EDTA, 0.5% taurocholic acid, 10mM β-mercaptoethanol). A 70 lb. stainless steel plate was placed overthe leaves/bags to assure complete immersion. A vacuum was pulled 27 in.Hg, held for 1 minute and then rapidly released. This vacuuminfiltration was repeated for a total of two cycles. Multipleapplications of the vacuum without isolating the intercellular fluidconstitutes a single infiltration procedure. An indication of completeinfiltration is a distinct darkening in color of the underside of theleaf tissue. Following the vacuum infiltrations, the leaves and basketwere removed from the vacuum tank. The bags containing the vacuuminfiltrated leaves were allowed to gravity drain surface buffer for ˜10minutes, prior to centrifugation. The intercellular fluid (IF) wasrecovered from the vacuum infiltrated leaves by centrifugation (1,800×g,30 minutes) using a Heine basket centrifuge (bowl dimensions, 28.0inches diameter×16.5 inches). Collected IF was filtered through a 50 uMcartridge filter and then stored at 4° C., until the entire 100kilograms of tissue was infiltrated. This process was repeated with thenext set of four 5 kg bags (5×20 Kg cycles total) until all the tissuewas infiltrated. Additional buffer was added during each infiltrationcycle to completely immerse the tissue.

Recombinant GCB was purified by loading the dilute intercellular (feedstream) directly on a Pharmacia Streamline 200 column containing PhenylStreamline resin. Expanded bed chromatography enabled us to capture,clarify and concentrate our protein in one step without the need forcentrifugation and/or microfiltration steps. The column was equilibratedand washed until UV-signal on recorder returned to baseline with 25 mMcitrate, 20% ethylene glycol, pH 5.0 and then eluted with 25 mM citrate,70% ethylene glycol. The eluted material was sterile filtered by passingthe eluted material through a 1 sq. ft. 0.8 um Sartoclean GF capsulefollowed by a 1 sq. ft. 0.2 um Sartobran P sterile filter (Sartorius,Corp.) and stored at 4° C. until the next chromatography step. Theeluted material from 4-5 days of Phenyl Streamline chromatography runswas pooled together and further purified on a cation exchange resin, SPBig Beads (Pharmacia), equilibrated in 25 mM citrate, 75 mM NaCl, pH5.0. GCB was eluted with a step gradient of 25 mM citrate, 0.4 M NaCl,10% ethylene glycol, pH 5.0. All chromatography steps were carried outat room temperature. The eluted material was sterile filtered by passingthe eluted material through a 1 sq. ft. 0.8 um Sartoclean GF capsulefollowed by a 1 sq. ft. 0.2 um Sartobran P sterile filter (Sartorius,Corp.) and stored at 4° C.

Using the suicide substrate, conduritol β-epoxide (CBE), inhibition ofrecombinant glucocerebrosidase (rGCB) activity in the presence of plantglucosidases was achieved. Enzyme activity was measured at 37° C. in areaction mixture containing 5 mM methylumbelliferyl β-D glucoside, 0.1 MPotassium Phosphate, 0.15% Triton-X100, 0.125% sodium taurocholate, 0.1%bovine serum albumin, pH 5.9 with and without CBE. Total glucosidaseactivity and rGCB activity were measured by hydrolysis of thefluorescent substrate 4-methylurnbelliferyl glucopyranoside. Totalprotein was determined using the Bio-Rad Protein Assay based on themethod of Bradford (Bradford, M. Anal. Biochem. 72:248; 1976). Table 7contains the GCB recovery data from TMV transfected plant tissue.

The quantity of virus present in IF extracted leaf tissue was determinedusing homogenization and polyethylene glycol precipitation methods. Inaddition, the amount of virus present in the pooled, intercellular fluidwas determined by direct polyethylene glycol precipitation. Final virusyields from precipitated samples was determined spectrophotometricallyby absorbance at 260 nm.

TABLE 8 Sample Virus Titer IF extracted leaf tissue 0.206 mg virus/gfresh weight Pooled IF 0.004 mg virus/g fresh weight, 0.010 mg virus/mlIF

EXAMPLE 10

Making rGAL-A Enzyme

Experimental Results. Achieving high steady-state mRNA levels is aprerequisite for vector development. However, there are many complexbiochemical and host compatibility interactions that ultimatelydetermine the overall performance of a heterologous expression systemfor a given protein. For this reason, we initiated some preliminaryexperiments to test the potential for RNA-viral mediated synthesis ofactive rGal-A in whole plants.

In order to ensure efficient delivery of rGal-A into the lumen of theplant endoplasmic reticulum, we fused the Gal-A cDNA (31) to a plantsignal peptide sequence derived from rice α-amylase gene (32,33). Wealso hypothesized that addition of an ER-retention signal (SEKDEL) (SEQID NO:37) might prolong the resident time of the recombinant protein inthe ER to increase the fraction of correctly assembled and catalyticallyactive enzyme under extreme conditions of protein synthesis. Theseconstructs were subcloned into the viral vector TTODA, a chimera betweentobacco and tomato mosaic viruses (FIG. 1). Transcripts were prepared invitro and inoculated onto the lower leaves of whole plants (Nicotianabenthamiana). 1-3 weeks after inoculation, leaves were weighed, rolledin a strip of Parafilm and placed in a disposable chromatography columnand submerged in enzyme extraction buffer (0.1 M K/P04, 0.1 M NaCl, 5 mMEDTA, 10 mM β-ME and 0.5% sodium taurocholate, pH 6.0). In order toinfiltrate the buffer into the tissue, a vacuum of 730-750 mmHg wastwice applied. After draining the excess buffer, the intercellular fluidfraction was recovered by low-speed centrifugation (˜1,500×g, 15 min).To measure enzyme remaining in the tissue after this treatment, the leafwas unrolled after centrifugation and two discs removed with a #14 corkborer. This tissue sample was transferred to an eppendorf tube, frozenin liquid nitrogen and ground in four volumes of enzyme extractionbuffer. In rGal-A enzyme assays, we measured cleavage of the fluorogenicsubstrate 4-methyl umbeliferyl α-D-galactopyranoside (4-MUG) againstknown standards using established protocols (34). Units are nmoles of4-MUG hydrolyzed per hour at 37° C.

In several initial experiments, plant leaves transfected with allconstructs accumulated 1-2% of the total soluble plant protein as crossreacting immunologic material (CRIM) using antisera specific for Gal-Ain quantitative Western analyses (data not shown). However, enzymeactivity was much lower than expected for this amount of CRIM andfurthermore was only 2-4 fold higher than activity due to endogenousplant α-galactosidase isozymes. It also appeared that addition of the ERretention signal allowed highest accumulation of steady state activityand that the IF contained little if any additional activity or CRIM.There are three cellular fates for any glycoprotein synthesized in aplant leaf: secretion to the IF, retention in the ER or sorting to thevacuole (35). We reasoned that because the ER retention signal slightlyincreased expression, the majority of the enzyme was inactivated laterin the secretory pathway. This could most likely occur by aggregation inthe trans-golgi network as is reported during over-production of thisenzyme in CHO-cells (36), and/or in the plant leaf vacuole. The IFfraction is quite clear and non-pigmented and is suitable for directchromatography. Using the initial construct (rGal-SEKDEL) (SEQ ID NO:24) we partially purified small amounts of rGal-A from the IF onhydrophobic, lectin and size exclusion resins.

For several plant proteins vacuolar sorting information is located in acarboxy-terminal propeptide (CTPP; 37,38). During the original cloningand characterization of human Gal-A, Quinn et al., postulated acathepsin-like potential CTPP cleavage for this enzyme at or near twoarginine residues, 26 and 28 AA from the termination codon (39,40). Theprecise AA sequence at the carboxy terminus has, to our knowledge, neverbeen reported. Because secretion in the plant leaf is through a defaultpathway we reasoned that deletion of specific sorting information from apostulated CTPP might yield more active enzyme in the IF. Analysis of asecond set of constructs containing either 12 or 25 AA truncations, withand without the ER retention signal provided dramatic evidence for thesignificance of this region (See Table 9). In one construct, rGal12-SEKDEL, virtually all of the CRIM is now assembled and stored asfully active enzyme and is secreted to the IF in significant quantities.As demonstrated in FIG. 3, rGal-A (˜52 kDa) is now the most abundantplant protein in a crude leaf IF sample. The other predominant band at17.5 kDa is the viral structural protein which likely contaminates thisfraction from broken trichomes of the leaf surface.

TABLE 9 rGal-A Expression (U/Gram Leaf Tissue) Intercellular ResidualContract/Sample Fluid Homogenate Total Experiment Uninfected Plant 2,800 7,500 10,300 rGal-A 5,100 10,900 25,000 rGal-A 5,400 15,000 20,400rGal-A-SEKDEL 6,800 30,300 37,100 rGal-A-SEKDEL 5,200 34,500 39,700Experiment Uninfected Plant 2,300  4,800  7,100 rGal-A 25 4,000  8,90012,900 rGal-A 25 2,300  9,000 11,300 rGal-A 25- 3,200 10,000 13,200SEKDEL rGal-A 25- 2,800  8,600 11,400 SEKDEL rGal-A 12 5,500 11,70017,200 rGal-A 12- 109,800  117,700  227,500  SEKDEL rGal-A 12- 199,000 329,500  528,500  SEKDEL

EXAMPLE 11

α-Galactosidase Expression Vector Development, Construction and Testing

The following example describes the series of α-galactosidase vectorsthat were constructed and tested for enzyme production. Initially, thehuman α-galactosidase A cDNA (Sugimoto, Y., Aksentijevich, I., Murray,G. J., Brady, R. O., Pastan, I., and Gottesman, M. M. Retroviralcoexpression of a multidrug resistance gene (MDRI) and humanα-galactosidase A for gene therapy of Fabry disease. Human Gene Therapy6:905, 1995.) was fused to a plant signal peptide sequence derived froma rice α-amylase gene (Kumagai, M. H., Shah, M., Terashima, M., Vrkljan,Z., Whitaker, J. R., and Rodriguez, R. L. Expression and secretion ofrice α-amylase by Saccharomyces cerevisiae. Gene 94:209, 1990.). Thischimeric gene was subcloned into the TMV based expression vector TTODAresulting in a construct designated rGAL-A, see Table 1. Vector rGAL-Awas modified by the addition of the putative ER retention signal SEKDEL,resulting in the vector designated rGAL-AR, see Table 1.

A series of C-terminal amino acid deletions were introduced into theα-galactosidase gene. Deletions of 4, 8, 12 or 25 codans from theC-terminus of rGAL-A were generated as well as the addition of theputative ER retention sequence (SEKDEL), see Table 10 and FIG. 12(sequence of TTODA (rGAL-12R). The deletion vectors were designated asdescribed in Table 10.

TABLE 10 Vector Designation Carboxy-Terminal Modifications (Amino AcidSequence) rGAL-A TSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 23) rGAL-ARTSRLRSHINPTGTVLLQLENTMQMSLKDLLSEKDEL (SEQ ID NO: 24) rGAL-4TSRLRSHINPTGTVLLQLENTMQMSL (SEQ ID NO: 25) rGAL-4RTSRLRSHINPTGTVLLQLENTMQMSLSEKDEL (SEQ ID NO: 26) rGAL-8TSRLRSHINPTGTVLLQLENTM (SEQ ID NO: 27) rGAL-8RTSRLRSHINPTGTVLLQLENTMSEKDEL (SEQ ID NO: 28) rGAL-12 TSRLRSHINPTGTVLLQL(SEQ ID NO: 29) rGAL-12R TSRLRSHINPTGTVLLQLSEKDEL (SEQ ID NO: 30)rGAL-25 TSRLR (SEQ ID NO: 31) rGAL-25R TSRLRSEKDEL (SEQ ID NO: 32)

The β-galactosidase gene fragment present in vector rGAL-12R was placedinto TMV vector SBS5. In addition, the rice β-amylase signal peptidepresent in rGAL-12R was replaced by the native human β-galactosidasesignal peptide. The resultant vector designated SBS5-rGAL-12R, see FIG.13, exhibited genetic stability upon serial passage on N. benthamianaplants.

α-galactosidase was extracted from inoculated plants using interstitialfluid and homogenization methods. Fluids were analyzed forα-galactosidase yield, enzyme activity and cellular partitioning andtargeting, see Table 11. In all cases, infectious transcripts wereprepared in vitro and inoculated onto the lower leaves of activelygrowing Nicotiana benthamiana plants. Characteristic viral symptoms,vein clearing and leaf curling, were noted ˜6-8 dpi (days postinoculation). Tissue samples were obtained from infected plants 1-3weeks after inoculation. Leaves were weighed, rolled in a strip ofParafilm and placed in a disposable chromatography column and submergedin extraction buffer (0.1 M K/P04, 0.1 M NaCl, 5 mM EDTA, 10 mM β-ME and0.5% sodium taurocholate, pH 6.0). Extraction buffer was infiltratedinto the tissue by pumping a vacuum of 730-750 mmHg. The vacuum wasapplied and released two times. After draining the excess buffer, theinterstitial fluid (IF) fraction was recovered by low-speedcentrifugation (˜1,500×g, 15 min). To measure enzyme remaining in thetissue after this treatment, the leaf was unrolled after centrifugationand two discs (˜1 mg each) removed with a #14 cork borer. This tissuesample was transferred to an Eppendorf tube, frozen in liquid nitrogenand ground in four volumes of extraction buffer. The total homogenatewas then subjected to centrifugation at ˜5,000×g and the supernatantfraction was saved for further analysis.

Extracts from IF and homogenates from post-IF leaf tissue were analyzedfor enzymatic activity by the hydrolysis of the fluorescent substrate4-methyl umbeliferyl α-D-galactopyranoside (4-MUG). Known standards andestablished protocols (Suzuki, K. Enzymatic diagnosis ofsphingolipidoses. Meth. Enzy. 138:727, 1987.) were used to obtain thenumber of enzymatic units (nmoles of 4-MUG hydrolyzed per hour at 37°C.) per gram fresh weight of tissue harvested.

TABLE 11 Interstitial Homogenate Total Enzyme Ratio of Vector FluidUnits/ Units/ Activity Activity IF/ Designation gram leaf gram leafUnits/gram leaf Homogenate Uninfected 3,837  9,404  13,241 0.41 rGAL-A6,833 189,971 196,804 0.04 rGAL-AR 6,829 312,068 318,897 0.02 rGAL-416,088  262,806 278,894 0.06 rGAL-4R 8,245 357,414 365,659 0.02 rGAL-8261,814  524,857 789,671 0.50 rGAL-8R 10,628  469,956 480,584 0.02rGAL-12 2,564  8,743  11,307 0.29 rGAL-12R 305,803  1,033,921  1,339,724   0.30 rGAL-25 1,265  6,629  7,894 0.19 rGAL-25R 2,489  6,394 8,883 0.39

Enzyme activity data from IF and homogenates derived from plantsexpressing α-galactosidase from the vectors in Table 2. indicate thatcarboxy-terminal deletions (4-12 codons) results in increasedx-galactosidase expression. Vector rGAL-12R expressed the highest levelof total α-galactosidase and also secreted the highest quantity ofactive enzyme.

EXAMPLE 12

Pilot Scale Purification of α-Galactosidase

Actively growing Nicotiana benthamiana plants, propagated in anuncontrolled horticultural greenhouse, were inoculated with encapsidatedtranscripts derived from the expression vector, SBS5-rGAL-12R. Tissuewas harvested 14-17 days post inoculation. Five kilograms of leaves wereplaced into polyester bags (Filtra-Spec®, 12-2-1053) and four×5 kg bagsof leaves were placed into a metal basket. The metal basket containingthe leaf material was placed in a 200 liter Mueller® vacuum tankcontaining ˜100 liters of buffered solution (50 mM acetate, 5 mM EDTA,10 mM 2-mercaptoethanol, pH 5.0). A 70 lb. stainless steel plate wasplaced over the leaves/bags to assure complete immersion. A vacuum waspumped to 695 mm Hg, held for 1 minute and then rapidly released. Thisvacuum infiltration was repeated for a total of two cycles. Multipleapplications of the vacuum without isolating the interstitial fluidconstitutes a single infiltration procedure. An indication of completeinfiltration is a distinct darkening in color of the underside of theleaf tissue. Following the vacuum infiltrations, the leaves and basketwere removed from the vacuum tank. The bags containing the vacuuminfiltrated leaves were allowed to gravity drain surface buffer for ˜10minutes, prior to centrifugation. The interstitial fluid (IF) wasrecovered from the vacuum infiltrated leaves by centrifugation (1,800×G,30 minutes) using a Heine® basket centrifuige (bowl dimensions, 28.0inches diameter×16.5 inches). The IF was filtered through a 50 μmcartridge filter to remove plant debris prior to purification.

Ammonium sulfate was added to the IF to 15% saturation, mixed for 10minutes and loaded onto a Pharmacia Streamline 200 column containing 4liters of Butyl Streamline resin equilibrated with 25 mM Imidizole, 15%(NH₄)₂SO₄, pH 6.0 at 1.2 L/min. The column was washed to UV baselinewith 25 mM Imidizole, pH 6.0, 15% (NH₄)₂SO₄ and α Gal was eluted with astep gradient of 25 mM Imidizole, pH 6.0. The eluent was filteredthrough a Sartorius glass fiber −>0.8 um cartridge filter and loadeddirectly onto 3 liters of Blue Sepharose in a Pharmacia BPG 200 columnequilibrated with 25 mM Imidizole, pH 6.0. The column was washed to UVbaseline with 25 mM Imidizole, pH 6.0 and α gal was eluted with a stepgradient of 25 mM Imidizole, 650 mM NaCl, pH 6.0. The eluent wasconcentrated using a 10 kD MWCO, cellulose acetate, 3 ft² spiralmembrane in an Amicon CH-2 concentrator and then sterile filtered.

Further purification was carried out either on Octyl Sepharose FF orHydroxyapatite. For Octyl Sepharose the column was equilibrated with 25mM Imidizole, 25% ammonium sulfate, pH 6.0 and eluted using a lineargradient of 25-0% (NH₄)₂SO₄ in 25 mM Imidizole, pH 6.0. ForHydroxyapatite purification, the sample was dialyzed overnight against10 mM KPO₄Buffer, pH 7.0 and then loaded on a column was equilibratedwith 10 mM KPO₄Buffer, pH 7.0. The column was washed with equilibrationbuffer until the UV reached baseline, followed by a linear gradient of10-200 mM KPO₄Buffer, pH 7.0. The α gal flowed through the column freeof the contaminating proteins.

Alpha gal activity was measured throughout the process with afluorescent assay using the synthetic substrate,4-methylumbelliferyl-α-D-galactopyranoside (MU-α gal). Enzyme activitywas measured at 37° C. in a reaction mixture containing 5 mMmethylumbelliferyl α-D-galactopyranoside, 0.1 M Potassium Phosphate,0.15% Triton-X100, 0.125% sodium taurocholate, and 0.1% bovine serumalbumin, pH 5.9. One unit of enzymatic activity hydrolyzes 1 nmol ofMU-α-gal per hour at 37° C. Total protein was determined using theBio-Rad Protein Assay based on the method of Bradford (Bradford, M.Anal. Biochem. 72: 248; 1976). Results of α-galactosidase activity(Total units and specific activity) from different enzyme productionlots are shown in Table 12.

TABLE 12 Specific Activity Units/mg protein Lot Number Kg BiomassExtracted Total Units (IF) (Purified) 981215   44.4 2.9 × 10⁹ 5.0 × 10⁶ 991115 100 5.5 × 10⁹ 3.6 × 10⁶  991116 120 6.9 × 10⁹ 4.0 × 10⁶  991117120 5.9 × 10⁹ 3.5 × 10^(6*) 991118   95.6 7.0 × 10⁹ 3.5 × 10^(6*) *Butyleluents from lots 991117 and 991118 were pooled before finalchromatography and purification. The data presented in Table 12demonstrates the consistency of pilot-scale extraction with respect tosecreted enzyme yield and specific activity.

EXAMPLE 13

Analysis of Purified α-Galactosidase

N-terminal Sequence Analysis

N-terminal sequence analysis of α-galactosidase, purified from plantsinoculated with transcripts derived from the vector rGAL-12R,MLDNGLARTPT (see SEQ ID NO: 1), had a 100% sequence homology to 11 aminoacids of human placental α-galactosidase with the addition of anN-terminal methionine. In contrast, N-terminal sequence ofα-galactosidase, purified from plants inoculated with transcriptsderived from the vector SBS5-rGAL-12R, LDNGLARTPT (see SEQ ID NO:2), wasas expected from native human enzyme. These data indicates the highdegree of fidelity that post-translational modifications are carried outwithin plant leaf cells and that human signal peptides are processedwith equal specificity in plants as in the native mammalian source.

C-Terminal Sequence Analysis

C-Terminal sequence of the rGAL-12R and SBS5-rGAL-12R plant producedenzyme was obtained by Edman degradation using the commercial service ofthe Mayo Foundation. Three cycles were achieved before the signal wastoo low to read additional sequence. Expected C-Terminus: LLQLSEKDEL(see SEQ ID NO:30).

Cycle Major amino acids 1st L, E 2nd D, V, A 3rd Q, G, T

It is important to note that the C-terminal amino acid was found to beheterogeneous, either L or E. The presence of glutamic acid in the firstcycle greatly reduced the signal because glutamic acid can form a cyclicstructure during the activation step that disables cleavage from thechain and therefore blocks a portion of the sample to furthersequencing. This reduced that ability of the software to interpret cycle3 and beyond. However, the presence of L, E and D in the first twocycles and the absence of other amino acids present in the analysis inan order resembling the α-galactosidase sequence strongly suggests thata population of the enzyme terminates with a DEL sequence as expectedfrom the sequence of the DNA clone.

Molecular Weight Determination

The apparent molecular weight of SBS5-rGAL-12R derived α-galactosidase(˜50 kDa) was quite similar to human α-galactosidase A, purified fromhuman placenta, as judged by both coomassie and silver stained SDS-PAGE.However, the protein purified from plant sources showed less molecularweight variation than the native human protein, indicating lessheterogeneity in plant glycosylation or a higher purity plant enzymepreparation.

The molecular mass of several lots of plant derived α-galactosidase weredetermined by MALDI-TOF mass spectroscopy to be 48,963, 48,913, 49,100daltons. These weights are consistent with the predicted mass ofα-galactosidase, based upon amino acid sequence, allowing for broaderpeaks due to glycosylation. The calculated molecular weight ofSBS5-rGAL-12R derived α-galactosidase is 44,619. The difference inpredicted and observed mass would equate to approximately 10.0%carbohydrate.

Glycan Analysis

There are four potential N-glycosylation consensus sequences (N—X—T/S)reported for human α gal A (Matsuura, et. al. Glycobiology 8:329-339,1998). We have identified four potential sites (108, 161, 184, 377) inour plant expressed α gal. One potential glycosylation site, in our αgal, is not glycosylated (377), as is the case for human α gal Aexpressed in CHO-cells.

Plants have both high mannose and complex glycans that differ frommammalian complex glycans by the presence of an α1,3 fucose on theproximal GlcNac and a β1,2 xylose on the β-linked mannose of the core.Four potential N-glycosylation sites have been identified for the plantderived α-galactosidase. The predicted amino acid sequence has fourpossible glycosylation sites (Asn—Xaa—Ser/Thr) at Asn residues (108,161, 184, 377). The glycosylation site at amino acid 377 was notglycosylated, similar to CHO cell derived α-galactosidase glycosylation.The four possible N-glycosylation sites are all located in β turnswithin hydrophilic regions of the enzyme. It was estimated the maturehuman α-galactosidase consists of about 370 amino acids and approx. 15%carbohydrate (Calhoun et al. PNAS 82: 7364-7368, 1985). Matsuura et al(Glycobiology 8:329-339, 1998) reports that in CHO-cell produced α galthere are four N-glycosylation sites (139, 193, 215, 407) and 3 of the 4sites are occupied (407 is not glycosylated).

We have determined that our plant expressed protein is indeedglycosylated because the enzyme will bind to ConA which has aspecificity for high mannose structures. Also, the plant derived enzymewas chemically deglycosylated with TFMS (trifluoromethanesulfonic acid).The α-galactosidase appeared to be cleaved as observed by a shift inmolecular weight on both a silver stained gel and a Western blot with αgal antibody. Early attempts to cleave rGal-A with PNGaseF to releaseN-linked carbohydrate have been unsuccessful suggesting the presence ofα1,3 fucose on the terminal GlcNac of the carbohydrate side chain. Thiswas verified by glycan analysis work carried out by the GlycobiologyCore Group at University of California San Diego Cancer Center.Carbohydrate profiling and compositional analysis was done. NMRexperiments confirmed that rGal-A from the plant IF contains an N-linkedglycan containing plant-specific carbohydrate linkages of a β1,2 xyloseand α1,3 fucose on the trimannosyl core. This N-linked structure hasbeen previously reported to occur in glycoproteins isolated from plantseeds and tissue cultures. Five (5) ug was hydrolyzed with 2M TFA for 4hours and analyzed by HPAEC-PAD. The total amount of sugar and sugarcontent was 560 ug and 12%. NMR analysis of the major peak showed atrimannosyl-chitobiose core, with α1,3 linked fucose and a β1,2 linkedxylose.

α-galactosidase glycan structures were determined by MALDI-TOF and/orMALDI-MS in collaboration with the Universitaet fuer Bodenkultur, seeTable 13. For MALDI, 5 μg of plant derived α-galactosidase was digestedwith pepsin in a mass ratio of 1:40 in 5% formic acid. After evaporationthe peptides were dissolved in ammonium acetate buffer, pH 5.0, boiledand subsequently digested with PNGase A overnight. Since the sample hasa mass of 49.000 g/mol, there are 100 pmol of glycoprotein. Afterevaporation, the peptides were removed by cation exchange chromatographyand the glycans are analyzed by MALDI (or pyridylaminated).

The molecular mass of the glycan was determined by MALDI-MS using aThermoBio Analysis DYNAMO (linear MALDI-TOF MS with delayed extraction)instrument. A small portion of the sample was dried on the sample targetand subsequently overlaid with “matrix” (gentisic acid). The samplescontained complex type sugar chains with fucose, xylose and varyingamounts of terminal GlcNAc. Small fractions were devoid of fucose andtherefore amenable to hydrolysis by PNGase F.

TABLE 13 Molecular Weight Lot #980805 Lot #981215 Glycan StructureDaltons % Glycan Structure % Glycan Structure MOXF 1050.5 3 — MMX 1066.52 2 MMXF 1212.7 22 8 Man 5 1237.5 — 1.8 GnMX/MGnX 1269.8 6 0.5GnMXF/MGnXF 1416.4 53 16 GnGnX 1473.3 5 7 GnGnXF 1619.9 9 55

TABLE 14 Characteristic Plant Derived CHO Cell Secreted Number of CoreStructures 8 23 Sialic Acid Absent Present Xylose β (1,2) Linkage AbsentFucose α (1,3) Linkage α (1,6) Linkage % Complex Structures % WeightGlycosylated 10-12% 15% Specific Activity

TABLE 15 Specific Activity α Galactosidase Source 4-MU SubstrateReference Nicotiana benthamiana  5.0 × 10⁶ This Patent ApplicationHuman, Recombinant Human Spleen 1.88 × 10⁶ Bishop and Desnick, 1981, J.Biol. Chem. 256 (3): 1307-1316 Human Placenta 0.99 × 10⁶ Bishop andDesnick, 1981, J. Biol. Chem. 256 (3): 1307-1316 Human Plasma 7.4 × 10⁵Bishop and Sweeley, 1978, Biochim. Bioph. Acta, 525:399-409

This example demonstrates the ability to extract two different productsfrom the same leaf tissue based upon extraction procedures thatspecifically target products localized in the apoplast and cytosol.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

All publications, patents, patent applications, and web sites are hereinincorporated by reference in their entirety to the same extent as ifeach individual patent, patent application, or web site was specificallyand individually indicated to be incorporated by reference in itsentirety.

Literature Cited

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1. An isolated polypeptide comprising the amino acid sequence of SEQ IDNO:
 18. 2. The isolated polypeptide according to claim 1, wherein saidpolypeptide is glycosylated at least at one amino acid position selectedfrom the group consisting of amino acid positions 108, 161, 184, and 337of SEQ ID NO:
 18. 3. The isolated polypeptide according to claim 1,wherein said polypeptide is glycosylated at least at one amino acidposition selected from the group consisting of amino acid positions 108,161, and 184 of SEQ ID NO:18.
 4. The isolated polypeptide according toclaim 1, wherein said polypeptide has a plant glycosylation pattern atamino acid positions 108, 161, and 184 of SEQ ID NO:18, wherein an alpha1,3 fucose is present on the proximal N-acetylglucosamine (GlcNac). 5.The isolated polypeptide according to claim 1, wherein said polypeptidehas a plant glycosylation pattern at amino acid positions 108, 161, and184 of SEQ ID NO:18, wherein a beta 1,2 xylose is present on abeta-linked mannose of the core.